Reaction-based selection for expression of and concentration of catalytic moieties

ABSTRACT

Disclosed and claimed are methods for selecting a recombinant virus, phage or cell expressing a catalytic antibody or catalytic portion thereof, or for selecting catalytic activity by a moiety. The method employs reaction-based selection for catalytic activity. The method can also be used to concentrate (increase the proportion of catalytic to non-catalytic moieties) a sample containing a catalytic moiety or viruses, phages or cells expressing a catalytic moiety. The selection or concentrating can be by employing a mechanism-based inhibitor, catalysis-accelerated movement, surface binding, changes in enthalpic component of binding as a function of temperature, or changes in binding by competition, or combinations thereof. The invention also comprehends a method for producing a recombinant virus or a cell-line expressing a catalytic moiety such as a catalytic antibody or catalytic portion thereof; and, this method can include infecting a suitable host with viruses which are screened for the expression. In addition, recombinant viruses and cell-lines so expressing a catalytic moiety such as a catalytic antibody or catalytic portion thereof are also disclosed and claimed.

This application is a divisional of U.S. Ser. No. 08/747,654, filed Nov.13, 1996, which is a divisional of U.S. Ser. No. 08/377,495, filed Jan.24, 1995, now U.S. Pat. No. 5,631,137, which is a continuation of U.S.Ser. No. 08/250,934, filed May 31, 1994, now abandoned, which is acontinuation of U.S. Ser. No. 08/101,274, filed Aug. 2, 1993, nowabandoned, which is continuation of U.S. Ser. No. 07/841,648, filed Feb.24, 1992, now abandoned.

FIELD OF THE INVENTION

This invention broadly relates to methods for selection for catalyticmoieties, to methods for increasing the concentration of catalyticmoieties in a sample containing catalytic moieties, and to substantiallypure or concentrated catalytic products from such methods. Thisinvention also relates to methods for selection or concentrating apopulation of recombinant viruses suspected of including virusesexpressing catalytic antibodies or catalytic portions thereof. Thisinvention also relates to detecting recombinant viruses that express acatalytic antibody or catalytic portions thereof and act catalytically.This invention further relates to a method for producing a recombinantvirus or a cell-line expressing a catalytic antibody or catalyticportions thereof by infecting a host susceptible to infection by arecombinant virus that expresses a catalytic antibody or catalyticportion thereof. This invention also relates to substantially purerecombinant virus or cell populations which express a catalytic antibodyor catalytic portion thereof from the aforesaid methods.

BACKGROUND OF THE INVENTION

Throughout this disclosure reference will be made to the publishedliterature by numerals in parenthesis. These numerical referencescorrespond to a listing of such literature references appearing at theend of this disclosure; all of these literature references being herebyincorporated herein by reference.

Viruses infect cells and divert the biosynthetic apparatus of the cellto synthesizing virus progeny. Certain viruses infect the host cell,cause the host DNA to break down and virus progeny to form in the cellwhereupon the cell lyses with the release of mature virus progeny. Otherviruses are lysogenic. These viruses infect a host cell and the viralDNA becomes inserted into a region of the host chromosome whereupon forgenerations the resultant cell-line (from replication of the infectedcell) expresses genetic products of the virus. A progeny of the originalinfected cell can spontaneously release the viral DNA from itschromosome or be induced to do the same, whereupon a lytic cycleresulting in viral progeny occurs. An example of this latter type ofvirus is the phage lambda which on certain occasions, e.g., exposure tocertain chemicals or radiation such as ultraviolet light, may initiate alytic cycle immediately after infection but can otherwise exist as aprovirus or prophage in the E. coli host genome for many generations.

A recent development in the field of antibodies is the amplification bythe polymerase chain reaction (PCR) of nucleotide sequences forantibodies or portions thereof (1). An extension of this development isthe insertion of these sequences into the genome of viruses, especiallyphages or bacteriophages (2, 3, 4, 11). In this regard reference isexpressly made to PCT Patent Publication WO920 1047, published Jan. 23,1992 entitled “Methods For Producing Members of Specific Binding Pairs,”incorporated herein by reference. Likewise, the expression of acatalytically-active enzyme on the surface of a phage has been achieved(32).

For instance, Clackson et al. (2) report using a random combinationallibrary of rearranged sequences for heavy (V_(h)) and kappa (V_(k))light chains from mice immune to the hapten 2-phenyloxazol-5-one (phOx)to display diverse libraries of antibody fragments on the surface of thefd phage. The recombinant fd phages were selected by passing thepopulation thereof over an affinity column.

Likewise, McCafferty et al. (3) report that complete antibody V domainscan be displayed on the surface of a recombinant fd bacteriophage andthat those that bind to an antigen (e.g., one in a million) can beisolated by affinity chromatography. And, McCafferty et al. (32) reportthe expression and affinity chromatography of functional alkalinephosphatase on the surface of a bacteriophage.

Similarly, Huse et al. (4) relate employing the bacteriophage lambdavector system to express in E. coli a combinatorial library of Fabfragments. Selection for expression was by selection for binding to anantigen.

A problem with the technique of selection of recombinant phagessuspected of expressing catalytic antibodies or catalytically activeportions thereof by hapten or antigen binding or affinity is thatinitially an enormous number of phages are produced; for instance, ofthe order of greater than 10⁵. Selection for hapten-binding from thisenormous population of phages still yields an enormous subpopulation ofphages (that bind); for instance, of the order of 6,000-10,000 phages.However, in this first subpopulation that bind there is yet a smallersecond subpopulation that not only express the antibody on their surface(and therefore bind to the hapten), but, also display a catalyticantibody (i.e., the antibody or portion thereof expressed is catalytic).Thus, isolation of only the first subpopulation (that bind with theantigen or hapten) does not adequately screen the recombinant phagepopulation to isolate those members which express the antibody orportion thereof catalytically. That is, hapten-binding selection isinsufficient to isolate those members of the recombinant phagepopulation which express catalytic antibodies or portions thereof forfurther use; e.g., for infecting a host cell such as E. coli andproducing consistent generations of recombinant phage or cellsexpressing the catalytic antibody or a catalytic portion thereof.Indeed, in a broader scope, a problem facing the development ofcatalysts such as catalytic antibodies, is the inability to economicallyenrich or select for moieties, e.g., antibodies, exhibiting the desiredcatalytic activity from among a vast excess of non-catalytic moieties,e.g., a vast excess of non-catalytic antibodies raised against the sametransition state analogs.

Further, prior methods for selection of catalytic activity of antibodyfragments (as opposed to their identification through extensiveselection exercises) depends on biological selection based on theability to compliment genetic defect in an organism expressing thefragment (16).

Heretofore there has been no method for selection of recombinant virusesor cells infected by such viruses displaying catalytic antibodies orcatalytic portions thereof based upon catalytic properties of suchviruses or cells.

In the area of enzymology the literature (5, 6) reports reactants calledmechanism-based inhibitors (affinity labels or suicide substrates).These reactants bind in the active site of an enzyme as normalsubstrates do, but, contrary to normal substrates, exploit the chemicalfeatures of the reaction mechanism to form an irreversible adduct withthe enzyme. Such reactants have been specifically designed for manyenzymes and enzyme mechanisms. Generally, a nucleophilic enzyme aminoacid residue that participates in the normal substrate catalyticreaction reacts instead with the mechanism-based inhibitor and ispermanently inactivated. Haptens which were suicide substrates have beenused to elicit antibodies (14). The suicide substrates were not used forselection of antibodies having catalytic activity.

Thus, heretofore there has been no application of mechanism-basedinhibitors to select recombinant phage or recombinant phage infectedcell populations for members expressing a catalytic antibody orcatalytic portion thereof or to increase the concentration of membersexpressing catalytic moieties. Nor has there been any application ofmechanism-based inhibitors to screen for catalytic moieties, such ascatalytic moieties expressed by phages, cells, or other self-replicatingsystems, or catalytic peptides, oligopeptides, polypeptides, or enzymes.Nor has there been any application of mechanism-based inhibitors toincrease the concentration of catalytic moieties in a sample containingcatalytic moieties.

Work with enzymes show that active enzymes can “crawl” across atwo-dimensional surface covered with substrate (on a micrometer distancescale), while inactive enzymes with the same binding affinity for thesubstrate are greatly restricted in their mobility (7, 8, 9). However,heretofore there has been no application of a two dimensional surfaceincluding a substrate of a desired catalytic reaction for selection ofrecombinant phage or recombinant phage infected cell population formembers expressing a catalytic antibody or catalytic portion thereof orto increase the concentration of members expressing catalytic moieties.Nor has there been any application of catalysis-accelerated movement toselect for catalytic moieties, or to increase the concentration ofcatalytic moieties in a sample containing catalytic moieties.

The kinetics of antibody binding to solid-phase immobilized antigen havebeen investigated (10). However, non-catalytic moieties have not beenseparated from catalytic moieties on the basis of surface binding. Forinstance, recombinant phages or recombinant phage infected cells havenot been screened or concentrated on the basis of those which express anon-catalytic antibody binding to the substrate with the same affinity,regardless of incubation time, whereas those which express a catalyticantibody or catalytic portion thereof initially binding to thesubstrate, but dissociating once catalysis has occurred. Nor has surfacebinding been employed to select for catalytic moieties in a samplecontaining catalytic moieties.

In addition, while effects of temperature on binding and catalysis byenzymes has been investigated (12), heretofore there has been no use ofthe discontinuity in the substrate binding of a catalytic moiety (butnot of a non-catalyst) as a function of temperature to select forcatalytic moieties, increase the concentration of catalytic moieties ina sample containing catalytic moieties or to screen or concentraterecombinant phages or recombinant phage infected cells expressingcatalytic moieties.

While principles of “weak affinity chromatography” in the presence of avariety of competing soluble ligands to alter the retention of moleculeson chromatographic columns, for instance of a ligand which possessesrelatively weak affinity for a moiety covalently coupled to a solidsupport (17) and catalytic mechanisms (15) have been examined,heretofore there has been no application of changes in binding ofcatalysts by competition to isolate or select catalysts fromnon-catalysts or to increase the concentration of catalysts.

It is desired to be able to select for catalytic moieties on the basisof those kinetic and thermodynamic properties intrinsically andessentially associated with catalysis, i.e., reaction-based selection isdesired. It is also desired to be able to increase the concentration ofcatalytic moieties in a sample, and to obtain this increasedconcentration by exploiting catalytic properties, i.e., obtaining anincreased concentration of catalytic moieties on a reaction basis. It isfurther desired to screen or concentrate a recombinant virus orrecombinant virus infected cell population which expresses a catalyticmoiety on the basis of catalytic properties, i.e., reaction basedselection or concentrating of such a population.

As mentioned earlier, it is also desired to be able to screen arecombinant phage population not only for those members expressing anantibody (e.g., by affinity or hapten binding), but, to also screen thispopulation for members which express a catalytic antibody or portionthereof, a catalytic enzyme, or more generally a catalytic moiety. Forinstance, selection based on catalytic properties is desired so thatthose members of the population which so express the catalytic antibodyor portion thereof can be used to produce further populations (withoutsubstantial contamination by members that do not express the antibody orportion thereof or that express it but not catalytically), or tocatalyze desired reactions (with optimal turnover rate due to minimalcontamination by or reduced concentration of members that do not expressthe catalytic antibody or portion thereof). Indeed, in the scenario ofattempting to use recombinant phages or the products of recombinantphage infected cells to catalyze a reaction, those members of thepopulation that express the antibody or portion thereof, but not in acatalytically active form, are deleterious to the reaction systembecause they can compete with catalytic phages or moieties forsubstrate. Likewise, with respect to using a recombinant phagepopulation to infect cells and produce monoclonal antibodies, reactionbased selection of the population is desired to reduce the laborinvolved in otherwise reducing the population to a smaller population tofurther create monoclonal antibodies. Thus, it is desired to be able toperform reaction based selection or concentrating of a recombinant phageor recombinant phage infected cell population for catalytic activity.

SUMMARY OF THE INVENTION

The present invention therefore provides a method for selectingcatalytic moieties comprising reaction-based selection for moieties withcatalytic activity and isolating such moieties. The present inventionlikewise provides a method for increasing the concentration of catalyticmoieties in a sample comprising reaction-based selection for moietieswith catalytic activity and isolating such moieties. The selection canbe by methods employing a mechanism-based inhibitor,catalysis-accelerated movement, surface binding, discontinuity ofbinding as a function of temperature, or changes in binding bycompetition.

The invention further provides a method for selecting a recombinantvirus or a cell, e.g., a cell infected by recombinant virus, whichexpresses a catalytic moiety such as a catalytic antibody or catalyticportion thereof, or for increasing the concentration of such viruses orcells in a sample of viruses or cells comprising:

reaction-based selection for catalytic activity of a population ofrecombinant viruses or cells suspected of including viruses or cellsexpressing the catalytic moiety, and

isolating from the population a subpopulation which can actcatalytically.

Similarly, the invention provides a method for producing a recombinantvirus or a cell-line capable of expressing a catalytic moiety such as acatalytic antibody or catalytic portion thereof comprising:

reaction-based selection for catalytic activity of a population ofrecombinant viruses suspected of including viruses expressing thecatalytic moiety,

isolating from the population a subpopulation which can actcatalytically, and

infecting a host susceptible to infection by the recombinant virus withvirus of the subpopulation.

The reaction-based selection can be performed by employing amechanism-based inhibitor, catalysis-accelerated movement, surfacebinding, discontinuity of binding as a function of temperature, orchanges in binding by competition. These embodiments also contemplaterepetition. If desired, for instance, the reaction-based selection canbe repeated after the isolating step; and, this repetition can be by thesame technique (such as repeated catalysis-accelerated movement), or, bya different technique (e.g., catalysis-accelerated movement during afirst pass through the reaction-based selection with surface binding,mechanism-based inhibitor, discontinuity of binding as a function oftemperature or changes in binding by competition employed during asubsequent, repeated pass through the reaction based selection step).

The invention further provides a method for detecting a recombinantvirus or a cell which expresses a catalytic moiety (e.g., a catalyticantibody or catalytic portion thereof), or for increasing theconcentration of such viruses or cells in a sample which comprises:

selection of a population of recombinant viruses or cells suspected ofincluding viruses expressing the catalytic moiety for binding to aselected hapten,

isolating from the population a first subpopulation which can bind tothe selected hapten,

reaction-based selection of the first subpopulation for catalyticactivity, and

isolating from the first subpopulation a second subpopulation which canact catalytically.

Likewise, the invention provides a method for producing a recombinantvirus or a cell-line capable of expressing a catalytic moietycomprising:

selection of a population of recombinant viruses suspected of includingviruses expressing the catalytic moiety for binding to a selectedhapten,

isolating from the population a first subpopulation which can bind tothe selected hapten,

reaction-based selection of the first subpopulation for catalyticactivity,

isolating from the first subpopulation a second subpopulation which canact catalytically, and,

infecting a host susceptible to infection by the recombinant virus withvirus of the second subpopulation.

The selection for binding to a selected hapten can comprise passing thepopulation over an affinity column of immobilized hapten. In this regardthe isolating of the first subpopulation can comprise eluting thatportion of the population which bind to the affinity column.

The selection for catalytic activity can be by employing amechanism-based inhibitor; for instance, the selection can comprisecontacting the first subpopulation with a mechanism-based inhibitor soas to form a reaction mixture. And, the isolating of the secondsubpopulation can comprise passing the reaction mixture over the haptenaffinity column and collecting viruses or cells which do not bind to theimmobilized hapten. Contacting of the first subpopulation with themechanism-based inhibitor can also be performed by contacting the firstsubpopulation with inhibitor bound to particles, for instance, bypassing the first subpopulation over a column to which the inhibitor isbound by a cleavable group; and, isolating the second subpopulation cancomprise cleaving the inhibitor-virus (or cell) complexes from thecolumn, or separating the inhibitor-virus (or cell) particle complexes.

Alternatively, the selection for catalytic activity can be by employingcatalysis-accelerated movement; for instance, selection can comprisecontacting the first subpopulation with a surface including a desiredsubstrate of a catalytic reaction, wherein the contacting is at a firstpoint on the surface, and, after sufficient time for members of thefirst subpopulation having catalytic activity to move a distance to asecond point on the surface, detecting members of the firstsubpopulation at the second point. In this alternative, isolating thesecond population can comprise collecting the members of the firstsubpopulation from the second point on the surface.

In a further alternative embodiment, the selection for catalyticactivity can be by surface binding; for instance, selection can comprisecontacting the first subpopulation with a surface including a desiredsubstrate of a catalytic reaction to which members of the firstsubpopulation not having catalytic activity will bind and members of thefirst subpopulation having catalytic activity will bind and engage incatalysis, and after sufficient time from the contacting for the membersof the first subpopulation not having catalytic activity to approachequilibrium with the surface but less than the time from contacting formembers of the first subpopulation having catalytic activity to consumesaid substrate, washing the surface to remove therefrom members of thefirst subpopulation having catalytic activity; and, isolating the secondsubpopulation can then comprise collecting the wash. In this alternativeembodiment the selection for catalytic activity can further comprise,after contacting the first population with the surface, prior to washingthe surface to remove therefrom members of the first subpopulationhaving catalytic activity and after sufficient time from the contactingfor both members of the first population not having catalytic activityand members of the first subpopulation having catalytic activity to bindto the substrate but-less than the time from contacting for members ofsaid first subpopulation having catalytic activity to consume substrateor complete catalysis, washing the surface so as to remove any membersof said first subpopulation having low or no affinity to the substrate.

In yet a further alternative embodiment the reaction-based selection canbe by changes in the enthalpic component of binding as a function oftemperature. For instance, the selection can comprise contacting thefirst subpopulation with substrate at a first temperature, contactingthe first subpopulation with the substrate at a second temperature, saidsecond temperature being higher than the first temperature, andthereafter the isolating of the second subpopulation comprisescollecting those members of the first subpopulation upon which exhibitthe effect of temperature on apparent binding is different. In thisembodiment the substrate can be immobilized, for instance on a column,members of the first subpopulation which bind loosely or elute first andthose which bind tightly or elute later at the first temperature arecollected in fractions; the fractions are again contacted with thesubstrate at the second temperature and fractions again collected. Thefractions collected after the contacting at the second temperature arethen analyzed for relative concentration of members of the firstsubpopulation: the fraction(s) having the lowest concentration(s)contain the members of the first subpopulation with the greatestcatalytic activity because the effect of the temperature on apparentbinding is different. It is preferred that the first temperature be solow that binding will occur, but not catalysis.

And in even yet another alternative embodiment the selection forcatalytic activity can be by changes in binding by competition. Forinstance, the selection can comprise contacting the first subpopulationwith an immobilized non-reactive substrate analog, said contacting beingin the presence of mobile non-reactive substrate analog and collectingtherefrom a sample or samples which bind to the immobilized analog;contacting the sample or samples with the immobilized analog in thepresence of substrate, and collecting therefrom those members of thefirst subpopulation which exhibit different apparent binding than amajority of the members. For example, if the immobilized analog isnon-reactive substrate analog immobilized on an affinity chromatographycolumn, when the sample or samples are contacted with the immobilizedcolumn in the presence of substrate, those members of the firstsubpopulation which exhibit different apparent binding than a majorityof the members are fraction(s) which elute later or sooner than the mainpeak(s).

Again it is mentioned that the methods of the invention encompassrepetition of any one step prior to advancing to the next step. Forinstance, an initial population can be subjected to the selection forbinding to a selected hapten, and those that bind can be isolated andthe isolated portion again subjected to selection for binding to thehapten, and then those that bind in this second pass through the stepare isolated and used in the reaction-based selection step. Likewise, aninitial “first subpopulation” can be subjected to the reaction-basedselection for catalytic activity, a second subpopulation which can actcatalytically is isolated therefrom, this second subpopulation can againbe subjected to the reaction-based selection, and, from subjecting thesecond subpopulation to a second pass through the reaction-basedselection step, a second “second subpopulation” can be isolated.Repetition of steps can be used to further enrich and increase thecatalytic activity (expression) of the product of the process. Thesecond pass through reaction-based selection can be by the sameprocedure as the first pass through, or can be by any of the otherembodiments of the invention. In addition, the invention provides forfurther alternatives of the above-described embodiments.

Thus, the present invention also provides a recombinant virus or a cellpopulation capable of expressing a catalytic moiety such as a catalyticantibody or catalytic portion thereof which is produced by the foregoinginventive methods. Such a population is substantially pure insofar asthe foregoing methods eliminate most, if not all, viruses, cells ormoieties which do not act catalytically, or those which do not bind tothe hapten as well as those which do not act catalytically. In addition,the present invention provides a catalyst composition prepared by theforegoing inventive methods. Such a catalyst composition includes theaforementioned virus or cell population, as well as other catalyticmoieties; and, such a composition is likewise substantially pure becausethe foregoing inventive methods eliminate most, if not all non-catalyticmoieties, or those which do not bind to hapten as well as those whichare non-catalytic. The viruses in the invention can be phages such asM13 phage or fd phage and thus, the host can be bacteria such as E.coli.

BRIEF DESCRIPTION OF DRAWINGS

In the following Detailed Description, reference will be made to theaccompanying drawings which are hereby incorporated herein by referenceand which assist in illustrating the invention without necessarilylimiting the invention, wherein:

FIGS. 1 and 2 are two dimensional separation profiles with first adimension elution profile (vol.) with non-reactive analog portrayedvertically and second dimension elution (vol.) profile with substrateportrayed horizontally, and the small starred peaks marking elutionafter or before main peaks so as to indicate fractions containingcatalysts which can then be enriched.

DETAILED DESCRIPTION

It is assumed that the skilled artisan already appreciates techniquesfor amplifying and inserting coding for antibody (or portion thereof)expression into the viral genome (1, 2, 3, 4, 11) and thus obtaining aviral population suspected of including members capable of expressing acatalytic moiety; and, it is assumed that the skilled artisanappreciates that such recombinant viruses can be used to infect a hostcell such as E. coli and that the recombinant virus can exist as aprovirus or prophage in the host chromosome with the cell and itsprogeny expressing the catalytic moiety until a lytic cycle isinitiated. It is also assumed that the skilled artisan alreadyappreciates techniques for obtaining a sample containing catalyticmoieties. Thus, the following description starts from a population ofrecombinant viruses or a population of cells suspected of includingviruses or cells capable of expressing a catalytic moiety, a catalyticantibody or catalytic portion thereof; or, from a sample, the presenceof catalytic moieties therein is to be determined, selected orconcentrated.

“Catalytic antibody” and “catalytic portion thereof” (of a catalyticantibody) as used herein is a substance which is capable of changing therate of a chemical reaction, all other conditions (e.g., temperature,reactant/substrate concentration, etc.) being the same and which is notirreversibly altered by the chemical reaction and, therefore, is notconsumed in the reaction. It is also a substance which exhibits thecapability of converting multiple moles of reactant/substrate per moleof catalytic antibody; and which, from a mechanistic viewpoint, bindsthe reactant/substrate, effects the accelerated conversion of thereactant/substrate to the product and then releases the product; andwhich changes the rate of the chemical reaction without shifting theposition of the equilibrium. While the aforementioned definitions arecharacteristics of ideal catalysts, in practice, even the best ofcatalysts become inhibited or deactivated by contamination in thereaction system or as a result of chemical or physical destructionduring the reaction process. For reasons well-known in the art, the trueoperation of a catalyst may be obscured by components of the reactionsystem or by the condition of the reaction environment.

In addition, the term “catalytic moiety” is used herein for any moietythat acts catalytically, and, this expression without limitationincludes: a “catalytic antibody”; a “catalytic portion thereof” (of acatalytic antibody); a catalytic peptide, oligopeptide, polypeptide orenzyme or catalytic portion thereof; catalytic molecules expressed byself-replicating systems; entities that contain catalytic molecules orthe expression thereof in their genetic coding and may beself-replicating or may not necessarily be self-replicating such as baremRNA, ribosomes or polysomes to which a catalytic molecule is attached,for instance by the building action of a protein thereon; or the like.

The term “surface-bound” is used herein for any moiety which is bound,for instance, either by physical attractive forces or chemical bonding,to a surface. As used herein, catalytic moieties are preferablysurface-bound either directly to DNA or RNA that codes for the catalyticmoiety or to an envelope enclosing the DNA or RNA that codes for thecatalytic moiety. Such an envelope can include, but is not limited to,cell membranes, viral coats, or cell walls.

The art has adopted certain working definitions to express catalyticactivity. These expressions include: (1) k_(cat), or “turnover number”;(2) k_(cat)/k_(uncat), the “rate enhancement factor” and (3) K_(m), the“Michaelis constant”. Turnover indicates the number of molecules ofreactant/substrate which can be converted to product per mole ofcatalytic antibody per unit time. For example, if a molecule exhibits aturnover of 10³ of substrate per minute and the molecule maintains itscatalytic activity for 24 hours at room temperature and at its optimalpH, each molecule of catalyst would then make a total of 1.4×10⁶conversions, indicating its catalytic behavior. This total conversion isto be distinguished from the total conversion in a stoichiometricreaction, which will never exceed 1.0, no matter how long the reactionis carried out. The rate enhancement factor is a dimensionless numberwhich expresses the rate of reaction in the presence of catalyst to therate of reaction in the absence of catalyst, all other reactionconditions being equal. The Michaelis constant is an apparent bindingconstant. It is equal to the concentration of substrate at which thereaction velocity is one-half maximal ([S]=K_(m) at k_(cat)/2).

In accordance with the invention, an antibody can comprise purifiedimmunoglobulins (IgG, IgM, IgA, IgD or IgE) or antibody fragments, suchas, for example, Fab, F(ab′)₂, Fv, etc., of immunoglobulins. Catalyticantibodies include certain major categories. A first category includescatalytic antibodies which have been rationally designed, i.e.,antibodies elicited with an antigen introduced by specific immunizationagainst a target antigen or substrate. Such catalytic antibodies,processes for their preparation and their use are described in U.S. Pat.No. 4,888,281, issued Dec. 19, 1989, U.S. Pat. No. 4,792,446,issued Dec.20, 1988 and U.S. applications Ser. No. 064,239, filed Jun. 19, 1987,and Ser. No. 805,576, filed Dec. 10, 1991; the disclosures of all ofwhich are incorporated herein by reference. A second category ofcatalytic antibodies includes naturally occurring antibodies which areproduced by an animal's immune system to the animal's own cellularcomponent (self-antigen), as opposed to the first category of catalyticantibodies previously described. These “autoantibodies” are described inU.S. application Ser. No. 343,081, filed Apr. 25, 1989, the disclosureof which is incorporated herein by reference. The DNA coding for both ofthese types of catalytic antibodies or for catalytic fragments thereofcan be amplified and be inserted into the viral genome (s 1, 2, 3, 4). Athird category includes catalytic antibodies which are generated bymethods not involving immunization. For instance, catalytic antibodiescan be produced by the randomization of variable regions of previouslyelicited antibodies to generate antibodies with new specificities, butwhich were not elicited from immunization.

The term “chemical reaction” refers to a reaction wherein at least onereactant is converted to at least one product. Such chemical reactionsinclude chemical reactions which can be catalyzed by enzymes such as,for example, oxidoreductases, transferases, hydrolases, lyases,isomerases and ligases, as well as chemical reactions for which noenzymes are known such as, for example, oxidations, reductions,additions, condensations, eliminations, substitutions, cleavages andrearrangements. The chemical reaction can also be the cleavage of apeptide bond. Peptide bond as used herein refers to an amide bondlinking two adjacent amino acid residues and is generically representedby the following formula wherein the peptide bond is shown within thebox:

An amino acid consists of a carbon atom to which is bonded an aminogroup, a carboxyl group, a hydrogen atom and a distinctive groupreferred to as a “side chain” (R^(a) and R^(b) in the formula above).Amino acid as used herein includes but is not limited to the twentynaturally occurring amino acids which comprise the building blocks ofproteins. It is understood by those skilled in the art that when eitherof the adjacent amino acids is proline, the respective side chains R^(a)or R^(b) are bonded to the adjacent nitrogen atoms to form thecharacteristic 5-membered proline ring.

The term “substrate” is synonymous with the reactant in the chemicalreaction and can be any of a number of molecules and biomoleculesincluding but not limited to esters, peptides, proteins, phospholipids,carbohydrates (e.g., glycogen, glucose, etc.) and drugs (includingabused substances and drugs and prodrugs from exogenous sources). Inthis regard reference is made to copending applications Ser. Nos.07/740,501, filed Aug. 5, 1991 and Ser. No. 07/773,042, filed Oct. 10,1991, incorporated herein by reference. For instance, the substrate cancontain an ester bond or bonds, peptide bond or bonds. The substrate canalso be any proteinaceous molecule, such as, for example, a regulatoryprotein or a structural protein including but not limited to peptidehormones (e.g., insulin, growth hormone, secretin, etc.), peptideneurotransmitters, neuromodulators and neurohumoral factors (e.g., VIP,endorphins, enkephlins, bradykinins, substance P, etc.), tumor proteins(e.g., oncogene products, carcinoembryonic antigens, etc.), bacterialproteins and viral proteins including without limitation core and coatproteins (e.g., human immunodeficiency viral (HIV) gp 120 influenzaglycoproteins, etc.). The substrate can also be an antigen or aself-antigen, i.e., any antigen which the animal body makes using itsown genetic code. Thus, self-antigens are distinguished from foreignantigens (e.g., bacterial, viral antigens). The term “animal” as usedherein refers to any organism with an immune system and includesmammalian and non-mammalian animals.

I. Mechanism-based inhibition selection

A. An Overview of the Method

In a general overview of this embodiment of the invention, a virus orcell population potentially bearing a catalytic antibody or catalyticportion thereof, or a sample containing catalytic moieties ispreferably:

1. Passed over an affinity column of immobilized hapten. Those that bindare eluted, by for example a change in pH or by passing free hapten overthe column. This binding selection is optional.

2. a. Viruses, cells or catalytic moieties found to bind to the haptenaffinity column are contacted with, for instance, mixed and allowed toreact with a suitably designed mechanism-based inhibitor to form amixture.

2. b. Alternatively, the mechanism-based inhibitor can be bound toparticles such as a column, preferably by a cleavable group. Theviruses, cells or catalytic moieties are passed over the column, withthose that bind being eluted for further use (e.g., infection) bycleaving the inhibitor from the column, or, by passing a suitablesolution, for instance an acidic solution, over the column, to dissolvethe virus' or cell's coat and eluting the DNA therefrom, and thereafterusing the DNA to transfect host cells or produce recombinant cells orviruses.

3. In the scenario of alternative 2.a. Of this embodiment, after anappropriate length of time, the mixture of viruses, cells or catalyticmoieties is chromatographed or re-chromatographed on the hapten affinitycolumn. This time the viruses, cells or catalytic moieties that flowthrough (that do not bind to hapten) are collected. These viruses orcells are used to infect a suitable host such as E. coli or are inducedto undergo a lytic cycle and the viruses resulting therefrom, e.g., fromhost infection or lysis are scaled up for catalytic use or furtherselection for catalysis.

This embodiment, in more detail, is as follows:

B. Hasten Affinity Chromatography

In preferred embodiments, before catalytic selection by mechanism-basedinactivation, the antibody expressing viruses or cells or the catalyticmoieties are pre-selected, for instance, by hapten affinitychromatography. However, catalytic selection can be performed withoutfirst pre-selecting by hapten binding. In a preferred embodiment, thoseviruses, phages, cells or moieties (>6000) that bind hapten aresubmitted to reaction with a mechanism-based inhibitor. Affinitychromatography of viruses bearing antibodies using immobilized haptenhas been successfully carried out in the literature (2, 3, 4). In thecurrent invention, the first step in the selection of catalysts ispreferably to use binding such as hapten affinity chromatography. Forexample, if hydrolysis of the following ester substrate (II) is desiredthe following hapten (I) can preferably be used:

For the hapten (I), R is a linker to a conjugate protein duringimmunization (e.g., KLH or BSA) and to a solid matrix such as a columnduring affinity chromatography and for the substrate (II), R¹ is —H or—NO₂.

Likewise, for hydrolysis of the following esters (IIA) and (IIB), thefollowing haptens (IA) and (IB) can preferably be used:

For these structures, Me=methyl group; and, reference is made tocopending application Ser. No. 07/773,042, filed Oct. 10, 1991,incorporated herein by reference, which describes the production and useof these compounds.

Those viruses, cells or moieties that bind to the hapten are eluted by achange in pH or with free hapten (followed by dialysis to remove boundhapten) and are then subjected to the next step; namely, exposure tomechanism-based reactants).

C. Reaction with Mechanism-Based Inhibitors

In this step the isolated subpopulation that binds to the hapten iscontacted with a suitable mechanism-based inhibitor (affinity label orsuicide substrate). A suitable mechanism-based inhibitor forms anirreversible adduct with a catalytically active antibody or portionthereof, for instance, a catalytic moiety expressed on the outer surfaceof the virus, phage, or cell whereas such an inhibitor does not form anirreversible adduct with a virus or cell which expresses the antibody orportion thereof but not catalytically. Below are describedmechanism-based inhibitors of ester-hydrolyzing antibodies that may beexpected to arise from immunization with haptens I, IA, or IB. Othermechanism-based inhibitors are well known that inactivate reactionsother than ester hydrolysis and could be used to select catalyticmoieties, such as catalytic antibodies, capable of catalyzing otherreactions. Catalytic reactions for which exist mechanism-basedinhibitors include synthetase, peptidase, oxidation/reduction,β-lactamase, decarboxylation, aminotransferase, lyase, racemase, andhydroxylase reactions (5). Design and use of similar inhibitors could becarried out by one skilled in the art for these and other reactions.

For instance, with reference to the above-mentioned esterolyticreactions the following structures are mechanism-based inhibitors:

(i) Hydrolysis of ester substrate (II):

wherein R^(s) is linkage to particles which may be packed in a column orsuspended in a fluid (mobile) and x is a leaving group such as Br, I,OSO₂CH₃ or the like. The linkage, of course, need not be present if theinhibitor is used in a mobile form.

Thus, in the instance of a catalytic antibody elicited by hapten (I) (ora phage or cell or the like which produces such a catalytic antibody ora catalytic portion thereof) contacted under reaction conditions withsubstrate II, the following occurs:

That is, a complex forms between the substrate and the catalytic moiety,e.g., catalytic antibody, which results in products (i) and (ii) and theregeneration of the catalyst.

However, in the instance of inhibitors (III) to (XI), while a reactionof the inhibitor occurs when contacted under reaction conditions withthe catalytic antibody elicited by hapten (I), a portion thereof remainsbound to the catalyst, thereby preventing it from engaging in furthercatalysis (preventing regeneration) and thus allowing for its isolationaccording to the invention, as shown in the following Table I.

TABLE I Binding Site Inhibitor Reactive Product Specificity III

Michael Acceptor for, thiol alcohol, phenol residues can form enamine w/amino IV

Michael Acceptor for, thiol alcohol, phenol residues can form enaminew/amino V

Alkylating agent for thiol, alcohol, phenol, amino, and carboxylresidues VI

Alkylating agent for thiol, alcohol, phenol, amino, and carboxylresidues VII

Alkylating agent for thiol, alcohol, phenol, amino, and carboxylresidues VIII

Michael Acceptor for, thiol alcohol, phenol residues IX

Michael Acceptor for, thiol alcohol, phenol residues can form enaminew/amino X

Michael Acceptor for, thiol alcohol, phenol residues can form enaminew/amino XI

Michael Acceptor for, thiol alcohol, phenol residues can form enaminew/amino

(ii) Hydrolysis of ester substrate (IIA):

Likewise, for the 5-fluorouridine esterase reaction, i.e., thehydrolysis of compound (IIA) or (IIB) to yield 5-fluorouridine bycontacting the compound (IIA) or (IIB) with an appropriate catalystunder reaction conditions (e.g., a catalytic antibody elicited by hapten(IA) or (IB), or a phage or cell or the like so expressing thatcatalytic antibody or a catalytic portion thereof), the following TableII provides inhibitors of the catalyst; and the portion of the inhibitor(“reactive product”) which remains bound to the catalyst; x and R^(s)are as defined above.

TABLE II Inhibitor Product Reactive Product XII

XIII

XIV

XV

XVI

XVII

XVIII

XIX

XX

In the above esterolytic reactions the conditions are generally: pH 6.0to 8.0, typically 7.0, 10 to 100, such as 30 to 70, typically 50 mMHepes, 100 to 300, such as 100 to 200, typically 150 mM NaCl,temperature of 20° to 40° C. usually 30° C., and time of 0.5 to 3 hours.

D. Separation of Viruses or Moieties by Affinity sorting

Following incubation, the viruses or cells bearing the antibody orportion thereof and that are catalytic, i.e., that have reacted with themechanism-based inhibitor, no longer bind hapten; but, those phages orcells bearing antibodies that did not react with the reactant willcontinue to bind to hapten. Thus, a separation of viruses or cellsdisplaying the catalytic antibody or the catalytic portion thereof fromnon-catalytic viruses or cells can be carried out definitely by a secondround of hapten affinity chromatography. This is carried out asdescribed above, and does not require separation of the viruses or cellsfrom unreacted mechanism-based inhibitor. Catalytic. antibody phages orcells do not bind to the column but non-catalytic phages do so bind.Thus, viruses or cells that pass through the column unbound are thephages which express the catalytic antibody or portion thereof. Theseviruses are used to infect a suitable host; for instance, if the virusesare phages such as M13, fd or lambda, E. coli is a suitable host. Afterinfection, the antibodies encoded in the viral DNA vector are scaled upfor further use or characterization. In particular, by so infecting E.coli with these phages, the E. coli can become a factory for theproduction of phages which express the catalytic antibody or portionthereof; or, for the expression of the catalytic antibody or catalyticportion thereof. The viral product or expression of the infected E. coliis then used catalytically, e.g., in ester hydrolysis, or, in furtherselection as herein described to produce virus (phage) and host (e.g.,E. coli) lines which are catalytically most active. Likewise, if cellshave been so catalytically screened and those that elute are collected,the cells can then be used to express the catalytic product; or, if thecells so express the catalytic product due to viral infection (with thevirus being a provirus or prophage), the cells can also be induced toundergo a lytic cycle.

Alternatively, the inhibitor is bound to particles. In a broad sense, acolumn is an example of particles, but they are “packed” (immobilized).The inhibitor is preferably bound to the particles through a cleavablegroup such that the reaction with the inhibitor is performed bycontacting the viruses, cells or catalytic moieties with the particles,e.g., passing them over the column. In the use of a column,non-catalysts elute, while catalysts bind; and, the catalysts areseparated by cleaving the inhibitor-catalyst complex from the column, orby otherwise causing disassociation from the inhibitor; for instance, inthe case of viruses, phages or cells, by passing a suitable liquid overthe column, e.g., an acid, and eluting the DNA therefrom for further use(transfection, recombination).

In particular, catalytic phages, viruses, cells or moieties selectedwith a specifically-designed mechanism-based inhibitor can be separatedfrom non-catalysts using affinity chromatography if the inhibitor isimmobilized to a solid matrix such as a column. In this embodiment it isdesired to be able to recover catalysts after they have reacted with theimmobilized inhibitor. To achieve this, the inhibitor can be attached tothe column via a molecule or molecules (a “cleavable group” or linkage)that can be readily cleaved or dissociated from the column matrix, toallow recovery of catalysts. The cleavable group can link the inhibitorto the column matrix either noncovalently or covalently.

In practice, a suspension of phages, viruses, cells or moieties, in asuitable buffer is eluted over a column on which an appropriateinhibitor has been immobilized via a cleavable group. The experimentalprocedure is depicted in the scheme below wherein on the left is anexample of a covalent cleavable group and on the right an example of anoncovalent dissociable linkage. Catalysts are able to react with theinhibitor and covalently attach to the column (support) via theinhibitor (center figure on both left and right) while those entitiesnot able to react with the inhibitor wash through the column (not shownin scheme). The column is then washed with approximately 5 columnvolumes of the initial elution buffer.

The buffer is chosen to optimize the reaction between the catalyst andthe inhibitor. Following the buffer wash, elution conditions are changedto cause cleavage (lower left in scheme) or dissociation (lower right inscheme) of the cleavable group to allow recovery of entities (catalysts)that have reacted with the inhibitor.

Noncovalent interactions provide a useful way of attaching an inhibitorto a solid matrix. The inhibitor is covalently attached to a ligandwhich is able to interact with the column in a dissociable noncovalentmanner. The column is a standard solid matrix such as Sepharose 6Baltered to display moieties that specifically and tightly interact withthe ligand attached to the inhibitor. (A general example would be theimmobilization on a column of an enzyme inhibitor. The inhibitor canreversibly and noncovalently bind to its corresponding enzyme whosesurface has been covalently modified to display one or more molecules ofsuicide substrate.) The interaction between modified column and ligandis of high affinity until the experimenter intentionally disrupts it bychanging the contents of the eluting solvent system (21, 22). The natureof the changes made in solvent system to elute the ligand are dependenton the choice of the noncovalently interacting pair of molecules, andcan be determined by the skilled artisan without undue experimentation.Examples of changes in elution conditions include: ligand competition,allosteric modification, substrate elution, inhibitor elution, ionicstrength changes, solvent changes to organic solvents or aqueous/organicmixtures, temperature changes, buffer or pH changes, metal elution,metal chelate elution, chaotropic reagents, or electrophoreticdesorption (23). Specific examples of interactions that can be used tononcovalently (but tightly) bind an inhibitor to a solid support arelisted below in Table III. See also Pierce, Amicon, Sigma Chem. Co.catalog incorporated herein by reference 24; 25, 26; 27):

TABLE III* Attached to solid support: Attached to suicide substrate:antigen (e.g., dinitrophenyl antibody group) antibody antigen cofactorenzyme enzyme cofactor protein A,G antibody carbohydrates, glycoproteinslectins lectins carbohydrate, glycoproteins heparin blood proteins blueA dye various proteins including: calmodulin serum albumin hormonereceptors renin red A dye various proteins including: alkalinephosphatase carboxypeptidase G peptide hormones matrix gel PBA (boronateRNA ligand) plasma proteins tRNA serine proteases iminobiotin avidinavidin iminobiotin immobilized metal α₂-macroglobulin carboxypeptidase A*(see catalogs of Pierce, Amicon, Sigma Chem Co. for immobilizedmatrices or instructions on immobilization)

Inhibitors can be linked to molecules listed in the righthand column bystandard methods such as carbodiimide coupling and others used inimmunology to couple haptens to keyhole limpet hemocyanin or bovineserum albumin.

As stated above, the elution conditions necessary to dissociate thenoncovalent complex will depend on the molecule pair chosen. Somespecific examples of eluents are listed below in Table IV (see also 21).

TABLE IV Associated Molecular Pair: Disruptive Solutes: proteinA/antibody acetic acid or glycine concanavalin A/oligosaccharideα-D-methylmannoside or borate buffer or α-D-methylglucoside blue Adye/albumin salt or urea immobilized EDTA or zinc/carboxypeptidase Ao-orthophenanthroline iminobiotin/avidin biotin or pH4 matrix gelPBA/trypsin boronate ion

An alternative to noncovalent cleavable interactions, the cleavablegroup can be covalently attached to both the column and the inhibitor.One way in which covalent cleavable groups can be disrupted is to makethem enzyme substrates. The group can then be cleaved by passing theappropriate enzyme through the column which will catalytically cleavethe group, releasing the inhibitor and attached catalysts. Perhaps themost versatile of examples of enzymatic cleavage is the use ofproteinase enzymes. The inhibitor and the column matrix can both becoupled to the protein substrate resulting in a “column—proteinasesubstrate—inhibitor” linkage where “—” represents a covalent bond.Exposure of the linkage to an appropriate protease hydrolyzes theproteinase substrate, releasing the inhibitor from the column allowingrecovery of attached catalysts. coupling procedures for attaching theproteinase substrate to the column and to the inhibitor are wellestablished using commercially available reagents (See. e.g. PierceChemical Co. catalog).

Specific examples of peptide covalently-attached cleavable groups andthe enzymes that can be used to cleave them are shown below in Table V.All proteinases and substrates listed are commercially available in apurified form (from Boehringer-Mannheim, for example):

TABLE V Proteinase Releasing Agent: Substrate Cleavable Group: ancrodfibrinogen elastase elastin casein ficin casein pepsin IgG hemoglobinsubtilisin casein thermolysin casein

Other covalent linkage groups may be cleaved using mild chemicalmethods. Table VI, below, lists some functionalized matrices, availablefrom commercial sources, which can be linked to inhibitors having athiol or amino group, and which can be detached by treatment withthiols. Table VI also lists linkage groups between the matrix andsubstrate required, and references to their use.

TABLE VI Matrix Linkage Reference Activated Thiol-Sepharose 4B (direct)(28) Activated Thiopropyl-Sepharose 6B (direct) (28) Mersalyl-Trisacryl(direct) (29) Avidin matrix NHS-SS-biotin (30) Avidin matrix HPDP-biotin(30)

Two protocols can be followed. In one protocol, the functionalizedsuicide substrate is reacted with the activated matrix ormatrix/linkage. The suspension of viruses, phages, cells or moieties isthen eluted through the column and allowed to react with the inhibitor.After washing out the noncovalently attached viruses, phages, cells ormoieties, the immobilized phages, viruses, cells or moieties can bedetached from the column by eluting with a thiol such asmercaptoethanol.

In the other protocol, the functionalized inhibitor is reacted with thesuspension of viruses, phages, cells or moieties. Phages, viruses, cellsor moieties with catalytic activity are covalently bound by theinhibitor. After an appropriate time, the suspension of viruses, phages,cells or moieties is eluted through the column of activated matrix ormatrix/linkage. Only those phages, viruses, cells or moieties havingcatalytic activity are retained via reaction of the thiol or amino groupof the inhibitor with the activated matrix-or linkage group. Theseviruses, phages, cells or moieties can then be detached by treatmentwith a thiol.

It is also noted that procedures akin to the foregoing for attaching aninhibitor to a column can also be employed for attaching an inhibitor tomobile particles, or for attaching non-reactive substrate analog orsubstrate to a column or membrane, as discussed below with respect tothe further embodiments of the invention.

Alternatively, the inhibitor can comprise or be bound to mobile(suspended) particles which are not “packed” or immobilized, i.e.,particles which are not in a column. In this instance, the viruses,cells or catalytic moieties are contacted with the particles. Again,catalysts bind to the particles, while non-catalysts do not. Thus, toseparate the catalysts from the non-catalysts, after contacting, theparticles are separated from the contacting medium, for instance, bygravity, filtration, centrifugation, electrophoresis, or by magneticfield (if the particles are magnetically responsive). After theparticles are isolated, it is preferred to wash them so as to remove anynon-catalysts which may have been included with the particles duringseparation.

The particles advantageously comprise micro-particulate matter having adiameter of 0.001 to 200 μm, such as 0.05 to 200 μm or 0.001 to 100 μm,preferably 0.1 μm to 100 μm, most preferably 0.5 μm to 10 μm, and asurface component capable of binding to the inhibitor. For example, themicroparticulate matter may be crosslinked starch, dextrans, cellulose,proteins, organic polymers, styrene copolymer such as styrene/butadienecopolymer, acrylonitrile/butadiene/styrene copolymer, vinylacetylacrylate copolymer, or vinyl chloride/acrylate copolymer, inertinorganic particles, chromium dioxide, oxides of iron, silica, silicamixtures, and proteinaceous matter, or mixtures thereof. A wide range ofparticles can be employed in the particle-based embodiments of theinvention. Generally the particles have a density of from 1.0 to 5.0g/mL and preferably have a density of from 1.1 to 2 g/mL. Choice of theoptimum density is within the skill of the art, the rate of settling ingravity-driven separation being a consideration.

Wide ranges of concentration of particles in the inhibitor compositioncan also be employed. For example, the concentration can range from 1 to10,000 μg/mL to preferably from 5 to 1000 μg/mL.

In the filtration mode of performing the invention, the filtration meansdesirably has a pore size, measured as mean diameter, from broadly 0.01to 90% of the mean diameter of the particles and preferably from 10% to90% of that diameter. The pore size of the filter should be such as toallow non-catalysts to pass therethrough, while preventing particles towhich catalysts bind from passing.

The art has described a number of magnetic particles which can be usedin the inhibitor compositions of the invention. For example, U.S. Pat.Nos. 4,628,037, 4,695,393, 4,698,302, 4,554,088, U.K. Patent ApplicationGB 2,005,019A and EP 0,180,384, all incorporated herein by reference,describe a variety of magnetic particles which can be used with success.The particles may be paramagnetic or ferromagnetic and may be coatedwith various materials to which inhibitor compounds are coupled so thatthe magnetic particle can be used in the invention. Desirably themagnetic particles used in the invention have a susceptibility of atleast 0.001 cgs units and desirably the susceptibility is at least 0.01cgs units. The magnetic particles may have a broad range of densities,i.e. from substantially less than that of water, 0.01, to 5 g/mL andpreferably from 0.5 to 2 g/mL. The particle sizes can range from 0.001to 200 such as 0.001 to 100 μm and preferably from 0.01 to 10 μm. Theconcentration of the particles may range broadly from 1 to 10,000 μg permL and preferably is from 5 to 1000 μg per mL.

Desirably, the magnetic particles which are used have a low magneticresonance, as described for example in EP 0,180,384, so that after themagnetic field is removed, the particles demagnetize. Desirably thedensity, concentration and particle size of the magnetic particles ischosen such that the settling time is at least 0.5 mm/min and desirablyit is above that rate. With respect to particles and particle-basedseparation methods, including electrochemiluminescence particle-basedseparation methods which can advantageously be used in the practice ofthis invention, reference is made to copending applications Ser. No.07/539,389, filed Jun. 18, 1990 and Ser. No. 07/652,427, filed Feb. 6,1991, both of which are incorporated herein by reference.

II. Selection By Catalysis-Accelerated Movement

A. An overview of the Method

Before catalytic selection the virus or phage or cell population orsample of catalytic moieties is preferably selected by hapten affinitychromatography. Those viruses, cells or moieties (>6000) that bind tohapten are preferably then submitted to catalytic selection.

This method involves a flat moist membrane or other two-dimensionalsurface which is covered, either through covalent attachment or not, bythe desired substrate of the catalytic reaction. The optimal surfacesubstrate concentration can be determined without undue experimentationby the skilled artisan for a particular substrate and membrane. Using asuitable instrument such as a very sharp instrument, a paste-likemixture of virons or cells potentially expressing the catalytic moiety,e.g., catalytic antibody, or a sample potentially containing a catalyticmoiety, is applied to, preferably streaked in a very fine line across,the surface (a first point on the surface). To visualize the streak asmall amount of dye, such as bromphenol blue, may be added to the paste.After a suitable length of time (typically 8 hours to three days but canbe otherwise depending upon the particular substrate, i.e, dependingupon the reaction being catalyzed and the conditions thereof; and can bedetermined without undue experimentation by the skilled artisan), thesurface of the membrane is scraped preferably with a sharp instrumentsuch as razor blade at a second point a suitable distance from the firstpoint, for instance 0.05 to 0.15 mm, typically 0.10 mm from the firstpoint (or line). To aid in the visualization of the small distance, thescraping can be carried out with the assistance of a low powermicroscope. Viruses or cells bearing catalytic moieties such ascatalytic antibodies, or more generally, catalytic moieties, are presentin the scrapings, having moved by catalysis-accelerated movement whileviruses or cells bearing non-catalytic antibodies or non-catalyticmoieties will not have so diffused. This process can be repeated toenrich the population or concentration of catalytic moiety expressingviruses, phages, or cells or more generally, of catalytic moieties,before scaling up for further use in catalysis or for use in producing anext phage generation (e.g., by infecting a host such as E. coli). Theprocess can also be enhanced by the use of electrophoresis. That is, themovement or diffusion of catalytic moieties or of viruses or cellsbearing catalytic moieties can be enhanced by applying a potentialgradient across the surface.

B. The Surface

The surface can consist of a number of various materials, agarose,starch, polyacrylamide, nylon, activated nylon, Immobilon AV (Millipore,Corp.), glass or nitrocellulose, for example. Important features in thisembodiment are that the surface should be able to support (covalently ornoncovalently) the substrate of choice, the viruses or phages or cellsor catalytic moieties should have some weak attraction for the surface,and it should be able to retain moisture without being immersed for thesuitable time periods, e.g., 8 hours to 3 days (possibly in anartificially-humid environment). The concentration of substrate on thesurface cannot be so small that the individual substrate molecules areseparated by large distances (>1000 angstroms). The surface is desirablycovered with a confluent monolayer of substrate wherein individualsubstrate molecules are separated preferably by no more than 500angstroms, more preferably by no more than 100 angstroms, and mostpreferably by no more than 50 angstroms. Concentrations of substrategreater than a confluent monolayer are acceptable and in some cases maybe preferable to a monolayer. One skilled in the art is able todetermine, without undue experimentation, based on weight, ultravioletabsorbance or other quantitation method, the concentration of substrateto be applied to the surface. If some other component in addition tocatalysis-accelerated movement is desired to assist in phage separation,such as an affinity chromatography component, the surface should becapable of serving in that capacity. For example, if phage movement isto be accelerated by electrophoresis across the substrate surface, thesurface should be capable of having an electric potential applied to it.

C. Application and Incubation of Viruses, Cells or Moieties ExpressingThe Catalyst

The viruses or phages or cells or catalytic moieties are applied to thesubstrate surface as a moist paste or very concentrated solutionpreferably using fine needled syringe. To be able to see where viruses,phages, cells or catalytic moieties have been applied, a small amount ofa dye such as bromphenol blue can be added to the sample beforeapplication. The phages or cells or catalytic moieties are preferablythen incubated on the surface in a suitable environment, e.g., ahumidity which is in excess of 50% or is “moist”; a suitable temperaturesuch as 20° to 40°, typically 25° or 37° C. for a time period dependingon conditions, which is typically between 8 hours and three days.

D. Selection of Viruses, Cells or

Moieties Expressing Catalytic Activity

Following incubation, the surface is preferably placed under a low powermicroscope or vision is otherwise assisted. A razor blade or other sharpinstrument is used to scrape the surface along a line on one or theother or both sides from the original point if it is substantially aline, approximately 0.05 to 0.15, typically 0.10 mm from the originalpoint; alternatively, if the original point was not appliedsubstantially as a line, the scraping can be along substantially acircumference or substantially a circle having a radius 0.05 to 0.15,typically 0.10 mm form the original point. Catalysts are more mobilethan non-catalysts and hence there is a concentration of catalysts inthe scrapings. This procedure can be repeated for further enrichment orconcentration of the catalytic phage or cell population (i.e., toproduce a population which is most catalytically active). Likewise, thisprocedure can, in general, be used to increase the concentration ofcatalytic moieties in a sample.

It may be necessary in certain situations to remove those viruses,phages, cells or moieties that do not bind to substrate, for instance,those which bind hapten but not substrate, since they may diffusereadily on the substrate surface. This is accomplished by, immediatelyupon streaking, quickly and lightly washing the surface or blotting thesurface with hapten. In this instance the viruses, phages, cells ormoieties which bind hapten but not substrate will initially diffusequicker than those which bind substrate (and are either catalytic ornon-catalytic); and thus, these substrate-non-binding hapten-binding andsubstrate-non-binding viruses, phages, cells or moieties can immediatelybe isolated and removed from the substrate surface. Non-catalystdiffusion is not a serious problem.

Likewise, in certain instances it may be desired to enhance diffusion byapplying a potential across the membrane surface, i.e., to enhancediffusion by electrophoresis. Typical conditions forelectrophoresis-enhanced movement are empirically determined by theskilled artisan, without undue experimentation, taking into account suchfactors as the medium (non-bound matter on surface, e.g., moisture onsurface as substrate has attraction to surface) the material beingdiffused (phage, cell, catalytic moiety), and the like, so that there isan optimum between the charge on the material and a minimizing ofconductivity of medium, and this promotes maximum separation. Thedistance for scraping (collecting catalysts) from the original point ofapplication to the surface is accordingly adjusted when movement bycatalysis is enhanced by electrophoresis.

III. Catalytic Selection By Surface Binding

A. Overview

This embodiment is a method to select for catalytic activity from amongan extremely large number of viruses, cells or moieties some displayingdifferent antibodies or moieties (e.g., catalytic versus non-catalytic).This method can be used for detection of catalytic activity, e.g., todetect expression of a catalytic moiety; or for increasing the catalyticmoiety concentration of a sample. It is based on the observation oftight-binding by a non-catalytic moiety such as an antibody which bindssubstrate and becomes immobilized on a membrane or other two-dimensionalsurface with the same affinity regardless of incubation time. On theother hand, a catalytic moiety such as a catalytic antibody or catalyticportion thereof initially binds to the substrate, but once catalysis hasoccurred it no longer binds to the solid surface. That is, aftersufficient time for non-catalysts to approach equilibrium with thesurface, but less than the time from contacting for catalysts to consumethe substrate on the surface, the non-catalysts remain bound to thesurface whereas catalysts can be washed therefrom (collected).

This catalytic selection embodiment preferably involves an initialselection for hapten binding moieties, e.g., antibodies, from weakly- ornon-hapten-binding moieties, e.g., antibodies. This can preferably bedone by the above-described procedures involving affinity chromatography(2, 3, 4) or filter-lift methods (31). Likewise, in other embodimentsherein, if an initial selection for hapten binding moieties isperformed, filter-lift methods may be used. Following selection of asubset of viruses, phages, cells or moieties by hapten binding, theviruses, phages, cells or moieties are then screened for substratecatalysis, i.e., reaction based or catalytic selection.

Catalytic selection in this embodiment preferably involvesimmobilization such as covalent immobilization of substrate on amembrane, such as Immobilon AV (Millipore Corp., Bedford, MA). To themembrane surface, a solution of viruses, phages, cells or moieties isadded. Alternatively, viruses, phages, cells or moieties can be directlytransferred by pressing the substrate-membrane against a sample such asinfected host, e.g., bacterial colonies, to lift associated virus orphage antibodies. Substantially immediately, the membrane is preferablywashed with a suitable buffer to quickly remove viruses, phages, cells,or moieties, e.g., antibodies, with a low or no affinity for thesubstrate. The washing step can be carried out by surface washing or byflow through of the buffered wash solution (buffered at a selected pH,e.g., 5.0 to 9.0 such as 7.0 and containing greater than 100 mM NaCl).Thereafter, the membrane is preferably washed with buffer again.Non-catalytic phages, viruses, cells or moieties, e.g., antibodies, thatbind tightly will not elute from the membrane, but viruses, phages,cells or moieties which are catalytic, e.g., viruses, phages or cellsbearing a catalytic antibody or catalytic portion thereof (or suchantibody or portion thereof lifted from infected colonies) that havecatalyzed enough substrate to become unbound, will elute. The wash canbe concentrated and used catalytically or to reinfect a host such as E.coli. Further enrichment of a phage, virus, or cell population bearing acatalytic antibody or catalytic portion thereof can be accomplished byrepeating the procedure. Likewise, the procedure can be used to increasethe catalytic moiety concentration of a sample.

In the procedure, the viruses, phages, cells (or antibody therefrom) ormoiety should remain in the local areas of the membrane with extremelylimited diffusion, such that catalytic phages, viruses or cells (orantibody therefrom) or catalytic moieties exhaust their local supply ofsubstrate and do not move a great distance to bind uncatalyzedsubstrate. Moreover, if diffusion takes place the catalytic phages,viruses or cells (or antibody therefrom) or catalytic moieties thencatalyze substrate in competition with binding, non-catalytic phages,viruses or cells (or antibody therefrom) or with non-catalytic moietiessuch that the non-catalysts will have a depleted substrate supply andwill also easily wash off the membrane. To prevent migration ofcatalytic moieties, e.g., catalytic phage, virus or cell moieties suchas antibodies, it is preferred to keep the membrane as free from excessmoisture, e.g., water, as possible and to only keep it moist; forinstance, in a humidified chamber. Also, the incubation times may bestbe minimized so as to prevent diffusion. Secondary washes to selectivelyremove catalysts can take place at suitable times such as intervalsbetween 0.5 to 16 hours, typically at 1 hour, 3 hours, 6 hours, and 12hours.

B. immobilized substrate

The substrate is preferably modified in the same way as the hapten inaffinity chromatography so that the substrate has a linker through whichit can be coupled to the membrane. This substrate linkage is akin tothat used to bind inhibitors to particles in the Mechanism BasedInhibition Selection embodiment of the invention, and vice versa. Thedetails of the chemistry thereof may depend on the particular substratechosen but can be closely based on the chemistry used in the haptenpreparation; for instance, if initial selection for hapten-bindingmoieties is performed, and is preferably very similar, if not identicalthereto.

C. Membrane

Substrate can be coupled via a linker to the membrane, for instance, anyof the surface materials described above with respect to selection bycatalysis accelerated movement, such as Immobilon-AV membrane. Thecoupling can be by known procedures for instance according to proceduressuch as the recommendations of the membrane supplier (Millipore) (see1990 Millipore catalog pp. 177-178, incorporated herein by reference);and likewise, the Selection By Catalysis-Accelerated Movement can employsuch linker procedures. The success of this embodiment, withoutnecessarily wishing to be bound by any one particular theory, may berelated to the concentration of substrate on the membrane (mmol/cm²) dueto steric limitations and other factors typically considered by thoseskilled in the art. The substrate concentration on the membrane can beas in the Selection By Catalysis-Accelerated Movement embodiment. Thus,the concentration of the substrate on the membrane can be optimizedwithout undue experimentation from this disclosure for any particularcatalytic moiety-substrate system.

Those skilled in the art will understand that other solid-phase surfacescan be substituted for the membrane. For example, non-magnetic ormagnetically responsive particles can be used as described above withrespect to selection by catalysis accelerated movement.

D. Application of Phage, Virus, Cell or Moiety

The phage, virus, cell or moiety binds to the membrane according toestablished binding kinetics of antibodies to antigens (10). Inaccordance with these findings, the exposure time of a phage-, virus-,cell- or moiety- containing solution to the substrate-membrane can be attwo minutes (rapid binders) and ten minutes (slow binders). After thesetimes, the membrane is preferably substantially immediately andpreferably quickly (for 1-10 preferably 1-5 seconds) given primary wash,either on the surface or by flow through. The wash is done to removemoieties, such as virus, phage or cell antibodies which bind weakly tosubstrate (e.g., extremely high apparent binding constant, K_(m),antibodies) or those non-specifically bound to the membrane surface.

E. Incubation

The slightly moist membrane can then be incubated at 20°-40°, typically25° or 37° C. in a humidified chamber for a suitable time such as 0.5 to16 hours typically 1 hour, 3 hours, 6 hours, or 12 hours, depending onthe particular moiety, e.g., antibody. A secondary wash is then carriedout for a suitable time typically 1-5 seconds in a chosen buffer(usually the same as the reaction buffer). More than one secondary washto remove catalysts can be done, although one wash is preferred as it ismuch less labor intensive. The secondary wash(es) can be done either onthe surface of the membrane or by flowing buffer through the membrane.

F. Use of the Secondary Wash

The secondary wash(es) containing the catalytic phages, viruses, cellsor moieties can be concentrated and prepared for catalytic use or forinfection of a suitable host e.g., E. coli with the phages, or forgeneration of catalysts by cells, e.g., causing recombinant virusinfected cells to undergo a lytic cycle. This membrane techniqueembodiment of the invention can be repeated with the secondary wash(es)to reduce the amount of any non-catalytic phages, viruses, or cells (orantibody therefrom) or of non-catalytic moieties which may have beenincluded in the secondary wash(es) and to further enrich theconcentration of catalytic moieties. Host, e.g., E. coli, coloniescontaining phage clones enriched for catalytic activity can be grown tosecrete sufficient catalyst, e.g., catalytic antibody or catalyticfragment thereof to be purified and screened or used directly forcatalytic activity. Purification of resultant catalysts, e.g., catalyticantibodies, can then be by standard procedures.

IV. Selection By Changes In Binding an a Function of Reaction ConditionSuch as Temperature or Competition

A. Overview Regarding Temperature

The effects of temperature on binding and catalysis differ for a givenprotein catalyst. Thus, as temperature increases from one temperaturesuch as a low or rather cold temperature at which there is binding butvery little catalysis to a second, generally higher temperature at whichthere is substantial catalysis, the apparent dissociation constant ofthe catalyst and its substrate will change in a discontinuous fashion.This change in apparent substrate binding of catalyst (but not anon-catalyst) with temperature change forms a basis for an embodiment ofthe invention for the separation, selection or concentration ofcatalytic moieties or of phages, viruses or cells capable of expressinga catalytic moiety such as a catalytic antibody or catalytic portionthereof from a large pool of non-catalytic moieties, phages, viruses orcells.

B. The Effect of Temperature on Binding and Catalysis Binding is subjectto the following physical observations and phenomena:${{E + S}\underset{k_{- 1}}{\overset{k_{+ 1}}{\rightleftarrows}}{ES}},{K_{s} = {k_{- 1}/k_{+ 1}}}$

ΔG=2.303 RT log K _(s)

where E is a catalyst, such as an enzyme or antibody, S is a substrate,ES is the Catalyst-Substrate complex (which can go to product, P), G isGibbs free energy, R is universal gas constant, it and T is thetemperature (in Kelvin).

Thus, a plot of T⁻¹ vs. log K_(s) for a binding molecule such as anenzyme at low temperature or a non-catalytic antibody will give astraight line with a slope of (2.303 R)/ΔG.

For a catalyst such as an enzyme or catalytic antibody, the apparentbinding constant is not K_(s) but K_(m). For the simple reaction shown:${{E + S}\underset{k_{- 1}}{\overset{k_{+ 1}}{\rightleftarrows}}{ES}}\overset{k_{+ 2}}{\rightarrow}{E + P}$

K _(m)=(k ₊₂ +k ⁻¹)/k ₊₁

The effect of temperature on k⁻¹/k₊₁(K_(s)) was discussed above. Theeffect of temperature k₊₂ is determined using the Eyring theory ofabsolute reaction rates. The effect of temperature on the reaction (k₊₂)is

log k ₊₂=[−(ΔH*+RT)/2.303]×T ⁻¹

(where ΔH* is the enthalpy of activation), so a plot of log k₊₂ (logV_(max)) vs. 1/T gives a slope of −(ΔH*+RT/2.303).

Thus, the effect of temperature on K_(m) depends on the relative sizesof k₊₂ and k⁻¹; the extreme cases being that K_(m)=k₊₂/k⁻¹ andK_(m)=K_(s). In most cases however, K_(m) equals some mixture of rateconstants (K_(m)=(k₊₂+k⁻¹)/k₊₁).

Combining the above concepts and observations, binding moieties such asantibodies have a linear relationship between 1/T and K_(s) since onlyone temperature coefficient is involved, whereas with catalytic moieties(such as an enzyme, catalytic antibody or catalytic portion thereof) theapparent binding constant (K_(m)) depends on two (or more)temperature-dependant phenomena, binding and catalysis, each having itsown temperature coefficient. Catalysis introduces into the enthalpicterm in the equation above, a factor that is temperature dependent.Therefore, in the case of a catalytic moiety, the relationship between1/T and K_(m) is not linear, but is curved depending on the differencesin catalysis and binding temperature coefficients. This embodiment ofthe invention exploits the effects of temperature on apparent binding toisolate, select, screen or increase the concentration of catalyticmoieties, such as viruses, phages or cells capable of expressing acatalytic moiety such as a catalytic antibody or catalytic portionthereof.

C. The Procedures, In General For Separation Based on Temperature

In general, in this embodiment a method is provided to separate phages,cells or moieties based on the effect of temperature on apparentsubstrate binding. Those that are catalytic respond differently totemperature changes than those that are non-catalytic. Moieties such asviruses, phage or cell antibodies having similar K_(s) values at lowtemperatures (where catalysis substantially does not occur) will havesimilar temperature dependencies of their K_(s) values. This is validfor the vast majority of moieties, e.g., viruses, phages or cells havingsimilar K_(s) values. In practice:

1. Moieties, e.g., viruses that bind hapten are separated on a column ofimmobilized substrate at a first, generally low temperature (e.g., −30to 0° C., typically −20° C., for example); those phages that bindloosely (having high K_(s)) elute first and those phages binding tightly(having low K_(s)) elute later.

2. Fractions are collected.

3. At a second, generally higher temperature (e.g., +20° to 45° C.,typically +25° or +37° C., for example), individual concentratedfractions are re-eluted on the same column and fractions once again becollected from each elution.

4. The fraction or fractions eluting containing the highestconcentration of viruses, phages, cells or moieties are detecteddirectly by absorbance or indirectly by Western blot, or in the case ofphages following infection of a host such as E. coli with an aliquot ofeach fraction. Viruses, phages, cells or moieties eluting in fractionsother than the main fraction(s) have different temperature coefficientsthan the viruses, phages, cells or moieties (non-catalytic) eluting inthe main fraction. Since they elute with different affinities from thebulk (noncatalytic) viruses, phages, cells or moieties, the, fractionseluting outside of the main fraction(s) can then be further enriched orconcentrated. The reason is that the viruses, phages, cells or moietieseluting outside the bulk fraction(s) have, in addition to a temperaturecoefficient for binding, a temperature coefficient for catalysis. Ingeneral, the effect of temperature on the apparent binding constant ofcatalytic viruses, phages, cells or moieties differ substantially fromthe bulk of non-catalytic viruses, phages, cells or moieties.

5. Enriched rare viruses or phages are then used to infect a host suchas E. coli and isolated moieties, therefrom, e.g., antibodies obtainedare used for catalysis or are further screened for catalytic activity.Likewise, enriched cells are isolated and used to express the catalyticmoiety or are induced to undergo a lytic cycle to produce a virus orphage generation. Similarly, enriched moieties isolated by the procedureare used for catalysis.

The above details of this embodiment of the invention can vary dependingupon the moiety and may involve substrate immobilized in a column matrixby standard methods (gravity or pump separation), on a electrophoresisgel (electric field separation), in a centrifuge tube (g-forceseparation), for instance a centrifuge tube containing immobilizedsubstrate suspended in a viscous solution, or other basic ways ofseparating viruses, phages, cells or moieties through suspended orimmobilized substrate. The low temperature separation is preferablycarried out in aqueous media, with the temperature kept low byrefrigeration, dry ice, or liquid nitrogen or liquid helium. The aqueousmedium is kept from freezing by inclusion of suitable concentrations ofNaCl or another salt or with glycerol. And, this embodiment can berepeated so as to further concentrate a sample of catalytic moieties,viruses, phages or cells.

D. Overview Of selection By Competition With Non-Reactive SubstrateAnalog

This embodiment employs an analog of the substrate in the catalyticreaction of interest. In the analog, the target bond is resistant toalteration by the catalytic species; but the analog has sufficientstructural similarity to exhibit similar binding affinity as thesubstrate molecule to the moiety, whether it be catalytic ornon-catalytic. An example of this for an esterolytic reaction of theR³COOR⁴ ester substrate is the structure R³COCH₂R⁴ which sufficientlymimics the ester structure, but the C(═O)CH₂ target bond whilestructurally similar to the ester is resistant to alteration by theesterolytic catalyst. A number of such nonreactive substrate analogsexist for any particular reaction and the selection of the appropriateanalog for use can be determined by the skilled artisan from thisdisclosure without undue experimentation, taking into consideration thestructural similarity between the potential nonreactive substrate analogand the substrate itself, the resistance to alteration of the targetbond in the analog, the chemical reaction involved, the binding affinityof the analog and other factors within the ambit of the skilled artisan.

Thus, this embodiment provides for separation or concentration ofcatalytic phages, viruses or cells or catalytic moieties by contacting asample thereof with a non-reactive substrate analog, preferably animmobilized analog such as an analog coupled to a solid phase supportpacked in a column. The contacting is preferably in the presence of suchan analog (mobile). From this contacting fractions are collected.Individually, fractions are again-contacted with the immobilizednon-reactive substrate, but in the presence of substrate. Fractions areagain collected. Catalytic phages, viruses or cells or catalyticmoieties dissociate from the substrate at a different rate than theirnon-catalytic counterparts such that if the analog is coupled to a solidphase support packed in a column, the catalysts elute outside of themain fraction, i.e., the catalysts elute either before or after the mainfraction.

In particular, virus, phage, cell or moiety in the sample which hassufficient catalytic activity (Ab) such that k₂>k⁻¹ in the reactionequilibrium equation (wherein “S” is substrate and “P” is product):${{Ab} + S}\underset{k_{-_{1}}}{\overset{k_{+_{1}}}{\rightleftarrows}}{{A\quad {bS}}\overset{k_{2}}{\rightarrow}{{Ab} + P}}$

elute in the second elution with retention times different than for themain peak of phages, viruses, cells or moieties eluting as the catalystsdissociate at different rates from the AbS complex than theirnon-catalytic counterparts. Collection of viruses, phages, cells ormoieties outside of the main peak will therefore enrich for (increaseconcentration of) or select for those exhibiting catalytic activity.Depending on the particular catalytic mechanism, catalysts elute in thesecond elution later or sooner than the main peak (15) see also FIGS. 1and 2; however, from this disclosure the skilled artisan can determinewhether the catalysts are eluting sooner or later than the mainfraction, without undue experimentation; for instance by determiningwhether that which elutes outside of the main peak is eluting before orafter the main peak, and by considering the turnover number achieved bythat which elutes outside of (before or after) the main peak, and bytaking into consideration other factors within the ambit of the skilledartisan. The noncatalyst association/dissociation with substrate (ornon-reactive substrate analog) is expressed as K_(s):

K _(s) =k ⁻ ₁ /k ₊ ₁

because the noncatalyst (Nc) associates and dissociates with substrateor nonreactive substrate (S*) only as follows:${{Nc} + S^{*}}\underset{k_{-_{1}}}{\overset{k_{+_{1}}}{\rightleftarrows}}{NcS}^{*}$

In the catalytic mechanism shown in the above scheme (with no catalyticintermediates occurring between the Michaelis complex (AbS) and productformation, P), the apparent association/dissociation of antibody andsubstrate can be written as K_(m):

K _(m)=(k ₂ +k ⁻ ₁ )/k ₊ ₁ .

Under these circumstances the value of K_(s) must always be less thanthat for K_(m); i.e., the non-catalytic viruses, phages, cells ormoieties bind substrate more tightly and hence elute later in the secondelution than catalytic viruses, phages, cells or moieties (see FIG. 1).

On the other hand, if the catalytic viruses, phages, cells or moieties(Ab) follow mechanisms in which reaction intermediates (such as covalentintermediates) occur on the reaction coordinate between the Michaeliscomplex and product formation, such as shown here in the mechanism below(wherein “S” is substrate, “AbS₂” is the intermediate, and “P” isproduct):${{{AB} + S}\underset{k_{-_{1}}}{\overset{k_{+_{1}}}{\rightleftarrows}}{A\quad {bS}}}\overset{k_{2}}{\rightarrow}{{A\quad {bS}_{2}}\overset{k_{3}}{\rightarrow}{{Ab} + P}}$

the term describing the apparent association/dissociation of substrateis different in this equation wherein K_(m) has a value that is lessthan that of K_(s), namely:

K _(m) =[K _(s) ][k ₃/(k₂ +k ₃)].

Thus, for this mechanism or any mechanism in which intermediates occurafter the AbS complex, the catalytic viruses, phages, cells or moietiesbind tighter and elute later in the second elution than thenon-catalysts (see FIG. 1).

However, if the reaction of AbS→Ab+P is very rapid such as to causeK_(s) to be greater than K_(m), or, if K_(s) for the NcS complex isgreater than K_(m), then the catalysts will elute sooner than thenon-catalysts, as shown in FIG. 2. Thus, collecting those moieties,cells, viruses or phages which exhibit different binding, e.g., elutelater or sooner, will not only be the isolation catalysts, but will alsobe the isolation of the best catalysts in the sample.

E. Particulars of Selection By Competition With Non-Reactive Analog

The non-reactive substrate analog is preferably coupled to a solid phasesupport to allow the preparation of an affinity matrix which ispreferably packed into a chromatography column. A suspension of viruses,phages, cells or moieties potentially including those which actcatalytically, e.g., phages, viruses or cells expressing a catalyticantibody or catalytic portion thereof which bind to a transition stateanalog used to generate the immune response which elicited the antibodyand the expression thereof, is applied to the column under conditionssuch as temperature, buffer composition, flow rate, which allow theviruses, phages, cells or moieties with an affinity for the substrateanalog to bind to the immobilized ligand such that they are retained onthe column relative to those with no affinity for the ligand. Theviruses, phages, cells or moieties eluting from the column at successivetime intervals are collected, pooled into appropriate fractions, andconcentrated. Inclusion of soluble non-reactive substrate analog in thebuffer used for the elution causes the binders in the sample to eluteearlier in the column profile by competing with the solid phase ligandfor binding to the viruses, phages, cells or moieties. Similarly,inclusion of the substrate in the eluting buffer has an identical effecton the elution of non-catalysts which bind to substrate with a similarK_(s) as they bind with the non-reactive substrate analog. Thus,fractions eluted with the non-reactive substrate analog from thenon-reactive substrate analog column are then again passed on thecolumn, but in the presence of substrate, preferably at equal or lowerconcentration than that at which the analog was used to achieve elution.

In the case of catalytic moieties, viruses, phages or cells, however thesubstrate will be either less or more effective at accelerating theirelution from the column because substrate is converted to product(s) bythe catalysts which are released from the binding site, allowing thecatalysts to re-bind to the immobilized ligand. Thus catalysts elutelater or earlier from a column eluted with substrate than they will fromthe same column eluted with non-reactive substrate analog. This formsthe basis of the two dimensional separation profiles shown in FIGS. 1and 2. In FIGS. 1 and 2 the first dimension elution with non-reactivesubstrate analog from the non-reactive substrate analog column isportrayed in the vertical direction and the second dimension elutionwith substrate from the non-reactive substrate analog column isportrayed in the horizontal direction. The starred peaks representfraction(s) eluting outside of the main fraction. The starred peaks arenormalized as shown in FIGS. 1 and 2 as the catalysts can elute as atrailing edge following the main peak (FIG. 1) or as a trailing edgepreceding the main peak (FIG. 2), i.e., the catalysts elutesignificantly later or earlier than the main peak(s) and are thus pooledto collect or concentrate the catalytically enriched population. FIG. 1shows the profile for K_(m)>K_(s) (catalysts binding longer thannon-catalysts), and FIG. 2 shows the profile for K_(s)>K_(m) (catalystseluting sooner than non-catalysts).

With respect to the hydrolysis of ester (II) (discussed above);

which ester can be generally shown by the following formula (XXXI)

wherein R₃ is

and R⁴ is

the following act

as a non-reactive substrate analog:

Thus, as shown herein, a non-reactive substrate analog for an ester canbe a ketone, carbonate, carbamate, ether such as a methylene ether, areversed ester, thio ester, imino ester, amide and reversed amide havingthe same or similar substituents (e.g., R³ and R⁴) as the substrateester. Of course, from this disclosure the skilled artisan can devisenon-reactive analogs for other substrates without undue experimentation.Moreover, non-reactive analogs can be devised for reactions other thanesterase reactions including peptide hydrolysis, oxidation/reduction,addition, elimination, condensation, isomerization, and transferreactions. Particularly since as shown herein a non-reactive analog isstructurally similar to the substrate, e.g., same or similar substituentgroups, but, the bond at the reaction site of the substrate is differentso as to resist alteration (reaction) by the catalyst but is similar tothe target bond nonetheless. For instance, if the target bond is a bondselected from ester such as typical ester, reversed ester, thio ester,or imino ester, ketone, carbonate, carbamate, ether such as typicalether or methylene ether, and amide such as typical amide, reversedamide and amino, the bond at the reactive site of the non-reactiveanalog is preferably another member selected from that group.

This embodiment can include further alternative embodiments. Forinstance, the coupling density of the ligand to the solid phase, thenature of the solid phase support, buffer composition, flow rate andconcentration of eluting species can be, from this disclosure, varied toachieve optimal selectivity (concentration or isolation of bestcatalysts) without undue experimentation, especially in view of theequations above. In addition, given the above equations, and the thrustof this embodiment for there to be competition in binding so thatcatalysts bind differently due to catalytic behavior and therefore elutedifferently, this embodiment and this disclosure thereof allows theskilled artisan, without undue experimentation, to select as compoundsfor the solid phase ligand(s) and eluting species not only theabove-described non-reactive analog and substrate, but also othercompounds such as transition state analog(s) and even unrelatedcompound(s) with significant affinity for binding to the moiety ofinterest. It is noted that Selection By Changes in Binding ByCompetition not only allows for isolation or concentration of catalysts;but, by the teachings herein, allows for separation of the bettercatalysts, especially since the selection is based upon and directlycorrelated to catalysis (K_(m)).

In a further alternative of this embodiment of the invention, thenon-reactive analog is attached to a two-dimensional support, such as amembrane as described above, and, the sample is driven across themembrane surface by forces other than gravity (which is used in thecolumn). For example, the sample can be driven across thenon-reactive-analog membrane (preferably moistened) by an electricfield, i.e., the separation of catalysts and non-catalysts iselectrophoretically induced. In this instance, the catalysts are againfound in the minority fraction. The conditions for electrophoresisinduced separation are empirically determined by the skilled artisanwithout undue experimentation by taking into account typical factors, asdiscussed above, so that there is an optimum between the charge on thesample undergoing separation and minimum conductivity of the medium(non-bound matter on membrane surface such as the moistening solutionthereon), as this promotes maximum separation by charge.

Of course, a catalytic population or sample (sometimes herein called a“subpopulation” or “second population”) of viruses, phages, cells ormoieties can be obtained by one method of this invention and either itor its next generation (such as from host infection or from inducing alytic cycle) can be further selected or concentrated by another methodof this invention. For instance, the secondary wash(es) (subpopulationor second subpopulation) from Catalytic Selection By Surface Binding canthen be subjected to Selection By Catalysis-Accelerated Movement,Selection By Changes in Enthalpic Component of Binding as a Function ofTemperature, Selection By Changes in Binding By Competition, orMechanism-Based Inhibitor Selection. Likewise, the scrapings(subpopulation or second subpopulation) from Selection ByCatalysis-Accelerated Movement can then be subjected to Selection BySurface Binding, Selection By Changes in Enthalpic Component of Bindingas a Function of Temperature, Selection By Changes in Binding ByCompetition, or Mechanism-Based Inhibitor Selection. Similarly, thesample(s) exhibiting a different effect of temperature on apparentbinding in Selection By Changes in Enthalpic Component of Binding as aFunction of Temperature (e.g., those that elute or bind outside of mainfraction(s)) can then be subjected to Selection By Surface Binding,Selection By Catalysis-Accelerated Movement, Selection By Changes inBinding By Competition, or Mechanism-Based Inhibitor Selection. And, thesample(s) exhibiting different binding when contacted with immobilizednon-reactive substrate analog in the presence of substrate (e.g., thosethat elute or bind outside of main fraction(s)) can then be subjected toSelection By Surface Binding, Selection By Catalysis-AcceleratedMovement, Selection By Changes in Enthalpic Component as a Function ofTemperature, or Mechanism-Based Inhibitor Selection.

The inhibitors, substrates and non-reactive substrate analogs discussedherein can be synthesized by the skilled artisan, without undueexperimentation, by following teachings in the art (see. e.g., 33).

The following non-limited Examples are given by way of illustration andare not to be considered a limitation of this invention, many apparentvariations of which are possible without departing from the spirit orscope thereof. In all cases these include reactions other than esterhydrolysis reactions, such as peptide hydrolysis, oxidation/reduction,addition, elimination, condensation, isomerization, and transferreactions.

EXAMPLES Example 1

Construction of Recombinant Phage Library

Generation of a library of VH and VL antibody domains for expression onthe surface of fd and M13 phages is accomplished using PCR technologyand appropriately designed consensus primers (1,2,3,4,11; see also PCTPublication WO920 1047 published Jan. 23, 1992 incorporated herein byreference). The starting material for PCR is spleen RNA obtained from amouse, immunized with hapten (I). A set of PCR consensus primers foramplification of mouse antibody variable domains is shown below and isalso as previously described (2), wherein SEQ A is provided to showpartial alignment of the primer sequence (SEQ ID NO: 1) and with codingfor the VH domain (SEQ ID NO: 2).

SEQ ID NO:1 5′end- 5′AGGTGAAACTGCAGGAGTCAGG 3′       CC G      C    TSEQ A SEQ ID NO:2 3′end- 5′TGAGGAGACGGTGACCGTGGTCCCTTGGCCCC 3′               VL EE SEQ ID NO:3 5′end- 5′GACATTGAGFCTCACCCAGTCTCCA3′SEQ ID NO:4 3′end- 5′CCGTTTGATTTCCAGCTTGGTGCC 3′  CCGTTTTATTTTCCAGCTTGGTCCC   CCGTTTTATTTCCAACTTTGTCCC  CCGTTTCAGCTCCAGCTTGGTCCC

Following amplification of VH and VL domains a second PCR reaction isused to link the domains together by a short peptide to produce singlechain Fv. Linking peptide, PCR primers and protocol are as previouslydescribed (2). Finally, a third PCR reaction is used to incorporateappropriate restriction enzyme sites at the 5′ and 3′ end of the singlechain Fv library to allow cloning into a phage display expression vectorsuch as fdDOG1 for fd surface display (2) or pComb3 for M13 surfacedisplay (11).

Alternatively, a variable domain library can also be obtained from anonimmunized mouse or other mammal such as a human. The startingmaterial in this instance is RNA from spleen (mouse or other mammal) orlymphocytes (human). Amplification and cloning is performed as describedabove except in the case of the human library the sequences of PCRconsensus primers are modified to reflect differences between mouse andhuman variable domains. Human consensus primers and construction of ahuman antibody phage display library is as has been previouslydescribed. (18).

Furthermore, the expression of Fab (as opposed to scFv) on the phagesurface, so that the phage library includes a Fab library, isaccomplished using a different set of 3′ PCR primers than those above soas to amplify those portions of the heavy and light constant regioncontaining cysteine residues involved in formation of disulfide bondsbetween the two chains; this is accomplished as previously described(4). Vectors for phage surface display of a Fab library include pHenIfor fd phage (19) and pComb3 (11) and pTacCP (20) for M13.

Using these techniques, recombinant phages expressing a catalyticantibody or catalytic portion thereof elicited by hapten I for the esterhydrolysis of substrate II are generated from fd and M13 phages; theinitial population of the recombinant phages is herein called cat1fd andcat1M13, respectively.

Example 2

Mechanism-Based Inhibitor Selection

A solution containing cat1fd is passed over an affinity column(Sepharose) containing compound of formula I as the bound hapten(linkage through R). A first subpopulation of phages (cat1fd1) that bindto the hapten are collected by subsequently passing free hapten over thecolumn followed by dialysis of phages eluted by the free hapten wash soas to remove bound hapten. This first subpopulation (cat1fd1) is testedfor its ability to catalyze the hydrolysis of the compound of formula II(R¹═NO₂) by determining the rate enhancement factor (pH 7.0, 50 Hepes,150 mM NaCl, 30° C., 0.5-3 hrs.); the first subpopulation shows a lowamount of catalytic activity.

A first portion of the first subpopulation (cat1fd1) is contacted withcompound of formula III (no R^(s) linkage) under the reaction conditionsfor hydrolysis of compound of formula II (pH 7.0, 50 mM Hepes, 150 mMNaCl, 30° C. 0.5-3 hrs.). This first portion is then passed over thehapten affinity column, and phages which in this pass do not bind to thecolumn are collected and used to infect E. coli. The resultant phagesfrom the E. coli infection (cat1fd2) are tested (rate enhancement) forthe ability to catalyze the hydrolysis of compound of formula II(R¹═NO₂) (pH 7.0, 50 mM Hepes, 150 um NaCl, 30° C., 0.5-3 hrs.); cat1fd2show greater catalytic activity (turnover number) than the firstsubpopulation. (cat1fd1).

A second portion of the first subpopulation (cat1fd1) is contacted withcompound of formula IV (no R^(s) linkage, x=Br) under the reactionconditions for hydrolysis of compound of formula II. This second portionis then also passed over the hapten affinity column, and phages which inthis pass do not bind to the column are collected and used to infect E.coli. The resultant phages from E. coli infection (cat1fd3) are tested(rate enhancement) for the ability to catalyze the hydrolysis ofcompound of formula II (R¹═NO₂) (pH 7.0, 50 mM Hepes, 150 mM NaCl, 30°C., 0.5-3 hrs.); cat1fd3 show greater catalytic activity (turnovernumber) than the first subpopulation (cat1fd1).

Example 3

Mechanism-Based Inhibitor Selection

Using the procedure of Example 2, third through tenth portions of thefirst subpopulation (cat1fd1) are each respectively contacted withcompound of formula V through XI (no R^(s) linkage, x=Br) under thereaction conditions for the hydrolysis of compound of formula II. Eachof the third through tenth portions is then passed over the haptenaffinity column (hapten I linkage through R) and those which do not bindare collected and used to infect E. coli. The resultant phages from E.coli infection (cat1fd4 through cat1fd10) are each tested (rateenhancement) for the ability to catalyze the hydrolysis of compound offormula II (R¹═NO₂) (pH 7.0, 50 mM Hepes, 150 mM NaCl, 30° C., 0.5-3hrs.); each of cat1fd4 through cat1fd10 show greater catalytic activity(turnover number) than the first subpopulation (cat1fd1).

Example 4

Mechanism-Based Inhibitor Selection

Using the procedure as in Example 2, a first subpopulation, cat1M131, isisolated from cat1M13 by hapten affinity chromatography. By determiningrate enhancement, cat2M131 shows a low amount of catalytic activity inthe hydrolysis of compound of formula II.

A first portion of cat1M131 is contacted with compound of formula III(no R^(s) linkage) under the reaction conditions for hydrolysis ofcompound of formula II. This first portion is then passed over thehapten affinity column and phages which in this pass do not bind to thecolumn are collected and used to infect E coli. The resultant phagesfrom E coli infection, cat1M132, are tested (rate enhancement) for theability to catalyze the hydrolysis of compound of formula II (R¹═NO₂)(pH 7.0, 50 mM Hepes, 150 mM NaCl, 30° C., 0.5-3 hrs.); cat1M132 showgreater catalytic activity (turnover number) than cat1M131.

A second portion of cat1M131 is contacted with compound of formula IV(no R^(s) linkage, x=Br) under the reaction conditions for hydrolysis ofcompound formula II. This second portion is then passed over the haptenaffinity column and phages which do not bind in this pass are collectedand used to infect E. coli.

The resultant phages from E. coli infection, cat1M133, are tested (rateenhancement) for the ability to catalyze the hydrolysis of compound offormula II (R¹═NO₂) (pH 7.0, 50 mM Hepes, 150 mM NaCl, 30° C., 0.5-3hrs.); cat1M133 show greater catalytic activity (turnover number) thancat1M131.

Example 5

Mechanism-Based Inhibitor Selection

Using the procedure of Example 4, third through tenth portions of thefirst subpopulation (cat1M131) are each respectively contacted withcompound of formula V through XI (no R^(s) linkage, x=Br) under thereaction conditions for the hydrolysis of compound of formula II. Eachof the third through tenth portions is then passed over the haptenaffinity column (hapten I, linkage through R) and those which do notbind are collected and used to infect E. coli. The resultant phages formE. coli infection (cat1M134 through cat1M1310) are each tested (rateenhancement) for the ability to catalyze the hydrolysis of compound offormula II (R¹═NO₂) (pH 7.0, 50 mM Hepes, 150 mM NaCl, 30° C., 0.5-3hrs.); each of cat1M134 through cat1M1310 show greater catalyticactivity (turnover number) than the first subpopulation (cat1M131).

Example 6

Selection By Catalysis-Accelerated Movement

A portion of the first subpopulation cat1fd1 from Example 2 is mixedwith bromphenol blue to make a phage paste. Compound of formula II(R¹═NO₂) is supported on Immobilon AV membrane (through R¹). A very fineline of phage paste is streaked across the compound of formulaII-Immobilon AV membrane. The membrane is incubated in a moist (50-100%relative humidity) environment at 25° C. for 48 hours. After incubation,with the assistance of a microscope, a razor is used to scrape andcollect phages 0.10 mm from the original line. The collected phages,cat1fd11, are tested (rate enhancement) for the ability to catalyze thehydrolysis of compound of formula II (R¹═NO₂) (pH 7.0, 50 mM Hepes, 150mM NaCl, 30° C. 0.5-3 hrs.); cat1fd11 show greater catalytic (turnovernumber) activity than cat1fd1.

Example 7

Selection By Catalysis-Accelerated Movement

Using the procedure in Example 4, the first subpopulation from Example6, cat1M131, is mixed with bromphenol blue to make a paste and a veryfine line of the paste is streaked across a compound of formulaII-Immobilon AV membrane. After incubation (50-100% rh, 25° C., 48hrs.), with vision aided by a microscope, a razor is used to scrape andcollect phages 0.10 mm from the original line. The collected phages,cat1M1311, are tested (rate enhancement) for the ability to catalyze thehydrolysis of compound of formula II (R¹═NO₂) (pH 7.0, 50 mM Hepes, 150mM NaCl, 30° C., 0.5-3 hrs.); cat1M1311 show greater catalytic activity(turnover number) than cat1M131.

Example 8

Selection By surface Binding

A portion of the first subpopulation cat1fd1 from Example 2 is added asa solution to a formula II-Immobilon AV membrane (Example 6) and aftertwo minutes and ten minutes the membrane is quickly washed (1-5 seconds)with buffer (same as solution). These washes are to remove any phageswhich are. weakly binding or non-binding to the substrate, and are thusdiscarded. The moist membrane is incubated (25° C., rh 50-100%) for 12hours with a quick 1-5 second wash (with buffer same as solution) at 1hour, 3 hours, 6 hours and 12 hours; these washes are collected,concentrated, and used to infect E. coli. The resultant phages from E.coli infection, cat1fd12 are tested (rate enhancement) for the abilityto catalyze the hydrolysis of compound of formula II (R¹═NO₂) (pH 7.0,50 mM Hepes, 150 mM NaCl, 30° C., 0.5-3 hrs.); cat1fd12 show greatercatalytic activity (turnover number) than cat1fd1.

Example 9

Selection By Surface Binding

A portion of the first subpopulation, cat1M131, from Example 4 is addedas a solution to a formula II-Immobilon AV membrane (Example 6) andafter two minutes and ten minutes the membrane is quickly washed (1-5seconds) with buffer (same as solution). These washes are discarded. Themoist membrane is incubated (25° C., rh 50-100%) for 12 hours with aquick 1-5 second wash (with buffer same as solution) at 1 hour, 3 hours,6 hours and 12 hours; these washes are collected, concentrated, and usedto infect E. coli. The resultant phages from E. coli infection,cat1M1312, are tested (rate enhancement) for the ability to catalyze thehydrolysis of compound of formula II (R²═NO₂) (pH 7.0, 50 mM Hepes, 150mM NaCl, 30° C., 0.5-3 hrs.); cat1M1312 show greater catalytic activity(turnover number) than cat1M131.

Example 10

Selection By Changes in Enthalpic Component of Binding as a Function ofTemperature

Cat1fd as in Example 1 are passed over a column (Sepharose) of compoundof formula II (R¹═NO₂, linkage through R¹) at −20° C. such that bindingcan occur, but not catalysis; and, fractions are collected. At 37° C.the fractions are again passed over the compound formula II column, withfractions again collected. Using absorbance, the fraction containing thelowest concentration of cat1fd (non-linear relationship between bindingand temperature) is isolated and used to infect E. coli. The resultantphages from E. coli infection, cat1fd13, are tested (rate enhancement)for the ability to catalyze the hydrolysis of compound of formula II(R¹═NO₂) (pH 7.0, 50 mM Hepes, 150 mM NaCl, 30° C., 0.5-3 hrs.);cat1fd13 show greater catalytic activity (turnover number) than cat1fd1.

Example 11

Selection By changes in Enthalpic Component of Binding As A Function OfTemperature

Cat1M13 as in Example 1 are passed over a column (Sepharose) of compoundof formula II (as in Example 14) at −20° C. such that binding can occur,but not catalysis; and, fractions are collected. At 37° C. the fractionsare again passed over the compound formula II column, with fractionsagain collected. Using absorbance, the fraction containing the lowestconcentration of cat1fd (non-linear relationship between binding andtemperature) is isolated and used to infect E. coli. The resultantphages from E. coli infection, cat1M1313 are tested (rate enhancement)for the ability to catalyze the hydrolysis of compound of formula II(R¹═NO₂) (pH 7.0, 50 mM Hepes, 150 mM NaCl, 30° C., 0.5-3 hrs.);cat1313.

Example 12

Selection By Changes in Binding By Competition

Cat1fd as in Example 1 are passed over a column (Sepharose) of compoundof formula XXI (linkage through R¹) and are eluted with a buffersolution containing compound of formula XXI. Fractions are collected andconcentrated. These fractions are then individually applied to the samecolumn and eluted with substrate (compound of formula II (R¹═NO₂). Thetwo dimensional separation profile is as shown in normalized FIG. 1: Thebulk of the phages eluting in the second pass over the column elute as amajor peak with about the same retention as for the first pass over thecolumn. The phages eluting later than the bulk are isolated and pooledand used to infect E. coli. The resultant phages from E. coli infection,cat1fd 14 are tested (rate enhancement) for the ability to catalyze thehydrolysis of compound of formula II (R¹═NO₂) (pH 7.0, 50 mM Hepes, 150mM NaCl, 30° C., 0.5-3 hrs). Cat1fd14 show greater catalytic activity(turnover number) than cat1fd1.

Example 13

Selection By Changes In Binding Competition

Using the procedure of Example 12, additional portions of cat1fd areeach respectively passed over a column. Each column is of compound offormula XXII through XXX and XXXII (linkage through R¹). Each portion iseluted with the respective non-reactive substrate analog linked to thecolumn. Fractions from each elution are then individually applied to thesame column from which they were eluted and this time eluted withsubstrate (compound of formula II, R¹═NO₂). The two dimensionalseparation profile for each is as shown in normalized FIG. 1. The latereluting phages from each elution are individually isolated and pooledand used to infect E. coli. The resultant phages from E. coli infection,cat1fd15 through cat1fd24, are tested (rate enhancement) for the abilityto catalyze the hydrolysis of compound of formula II (R¹═NO₂) (pH 7.0,50 mM Hepes, 150 mM NaCl, 30° C., 0.5-3hrs.); each of cat2fd15 throughcat1fd24 show greater catalytic activity (turnover number) than cat1fd1.

Example 14

Selection By Changes In Binding By Competition

Cat1M13 as in Example 1 are passed over a column (Sepharose) of formulaXXI (linkage through R¹) and are eluted with a buffer solutioncontaining compound of formula XXI. Fractions are collected andconcentrated. These fractions are then individually applied to the samecolumn and eluted with substrate (compound of formula II, R¹═NO₂. Thetwo dimensional separation profile is as shown in normalized FIG. 1: Thebulk of the phages eluting in the second pass over the column elute as amajor peak with about the same retention as for the first pass over thecolumn. The phages eluting later than the bulk are isolated and pooledand used to infect E coli. The resultant phages form E. coli infections,cat1M1314 are tested (rate enhancement) for the ability to catalyze thehydrolysis of compound of formula II (R¹═NO₂) (pH 7.0, 50 mM Hepes, 150mM NaCl, 30° C., 0.5-3 hrs.); each of cat1M1314 show greater catalyticactivity (turnover number) than cat1M131.

Example 15

Selection By Changes In Binding Competition

Using the procedure of Example 21, additional portions of cat1M13 areeach respectively passed over a column. Each column is of compound offormula XXII through XXX and XXXII (linkage through R¹). Each portion iseluted with the respective non-reactive substrate analog linked to thecolumn. Fractions from each elution are then individually applied to thesame column from which they were eluted and this time eluted withsubstrate (compound of formula II, R¹═NO₂). The two dimensionalseparation profile for each is as shown in normalized FIG. 1. The latereluting phages from each elution are individually isolated and pooledand used to infect E. coli. The resultant phages from E. coli infection,cat1M1315 through cat1M1323, are each tested (rate enhancement) for theability to catalyze the hydrolysis of compound of formula II (R¹═NO₂)(pH 7.0, 50 mM Hepes, 150 mM NaCl, 30° C., 0.5-3 hrs.); each ofcat1M1315 through cat1M1323 show greater catalytic activity (turnovernumber) than cat1M131.

Example 16

Construction of Phage Library

Using the procedures set forth in U.S. application Ser. No. 07/773,042,filed Oct. 10, 1991, mice are immunized with hapten IA or IB to elicitcatalytic antibodies which catalyze the hydrolysis of compound IIA orIIB to yield 5-fluorouridine.

Employing the procedures of Example 1, spleen RNA from the immunizedmice, PCR consensus primers for amplification, PCR to link domains by ashort peptide to produce single chain Fv, PCR to incorporate restrictionenzyme sites at the 5′ and 3′ end of the single chain Fv library, andcloning into respective phage display vectors of fd, lambda and M13 areemployed to produce recombinant phages expressing a catalytic antibodyor catalytic portion thereof elicited by hapten IA or IB for the esterhydrolysis of substrate IIA or IIB; the initial population of theserecombinant phages is herein called cat2fd, ca2λ and cat2M13respectively.

Example 17

Mechanism-Based Inhibitor Selection: Generation of First Subpopulation

A solution containing cat2fd is passed over an affinity column(Sepharose) containing compound of formula IA as the bound hapten(linkage through NH₂). A first subpopulation of phages (cat2fd1A) thatbind to the hapten are collected by subsequently passing free haptenover the column followed by dialysis of phages eluted by the free haptenwash so as to remove bound hapten. This first subpopulation (cat2fd1A)is tested for its ability to catalyze the hydrolysis of both compound offormula IIa and IIB by determining the rate enhancement factor (pH 7.0,50 mM Hepes, 150 mM NaCl, 30° C., 0.5-3 hrs.); the first subpopulationshows some catalytic activity. This procedure is repeated with anothersolution containing cat2fd, but the bound hapten is compound of formulaIB. The first subpopulation from the repeated procedure, cat2fd1B alsoshows some catalytic activity in the hydrolysis of both compound offormula IIA and IIB. Cat2fd1A and cat2fd1B are pooled for the firstsubpopulation, cat2fd1.

In the same fashion a first subpopulation from cat2M13, namely cat2M131,is generated. Cat2M131, as well as subsubpopulations from which they aregenerated (cat2M131A and cat2M131B) show some catalytic activity in thehydrolysis of both compound of formula IIA and IIB.

Example 18

Mechanism-Based Inhibitor Selection

A first portion of the first subpopulation cat2fd1 is contacted withcompound of formula XII covalently linked to a Sepharose column, asdescribed above, under the reaction conditions for hydrolysis ofcompound of formula IIA or IIB (pH 7.0, 50 mM Hepes, 150 mM NaCl, 30°C., 0.5-3 hrs.). That which elutes is collected and discarded. Mildinorganic acid (HCl) solution pH4-5) is passed over the column,and theDNA thereby eluted is used in E. coli transfection. The resultant phagesfrom the E. coli transfection (cat2fd2) are tested (rate enhancement)for the ability to catalyze the hydrolysis of both compound of formulaIIA and IIB (pH 7.0, 50 mM Hepes, 150 mM NaCl, 30° C., 0.5-3 hrs.);cat2fd2 show greater catalytic activity (turnover number) than the firstsubpopulation (cat2fd1).

Example 19

Mechanism-Based Inhibitor Selection.

Following the procedure of Example 18, a second subpopulation, cat2M132is generated from a portion of the first subpopulation cat2M131, and istested for catalytic activity. Cat2M132 show greater catalytic activitythan cat2M131.

Example 20

Mechanism-Based Inhibitor selection

Following the procedure of Example 18, except that compound of formulaXIII through XX are linked to the column, second subpopulations cat2fd3through cat2fd10, respectively (from use of compound of formula XIII-XX)are generated from portions of the first subpopulation cat2fd1, and aretested for catalytic activity. Each of cat2fd3 through cat2fd10 showgreater catalytic activity than cat2fd1.

Example 21

Mechanism-Based Inhibitor Selection

Following the procedure of Example 20, second subpopulations cat2133through cat2M1310 are generated from portions of the first subpopulationcat2M131 through the use of compound of formula XIII-XX, respectively;and, the second subpopulations are tested for catalytic activity. Eachof cat2M133 through cat2M1310 show greater catalytic activity thancat2M131.

The foregoing Examples demonstrate the surprising advantages of theembodiments of the present invention: Catalysts can be selected orconcentrated from non-catalysts, and better catalysts are obtained.

REFERENCES

1. Sastry, L., et al., Cloning of the Immunological Repertoire inEscherichia coli for Generation of Monoclonal Antibodies: Constructionof a Heavy Chain Variable Region-Specific CDNA Library, Proc. Natl.Acad. Sci., USA (1989) 86; 5728-32.

2. Clackson et al., Making Antibody Fragments Using Phage DisplayLibraries, Nature (1991), 3; 624-28.

3. McCafferty, J., et al., Phage Antibodies: Filamentous PhageDisplaying Antibody Variable Domains, Nature (1990), 3; 552-554.

4. Huse, W. D., et al., Generation of a Large Combinatorial Library ofthe Immunoglobulin Repertoire in Phage Lambda, Science (1989), 24;1275-81.

5. Silverman, R. B., Mechanism-Based Enzyme Inactivation:

Chemistry and Enzymology, Vols. I and II, CRC Press (1988) Boca Raton,Fla.

6. Shokat, K. and Schultz, P. G. lectures at the Ciba Symposium onCatalytic Antibodies, Sep.30-Oct. 3, 1990, London, England.

7. Henis, Y. I., et al., Mobility of Enzymes on Insoluble Substrates:The Beta-Amylase-Starch Gel System, Biopolymers (1988), 27; 123-138.

8. Katchalski-Katzir, E., et al., Enzyme Diffusion and Action on Solubleand Insoluble Substrate Biopolymers, Biopolymers (1985) 24; 257-277.

9. Richter, P. H. & Eiger, M., Diffusion Controlled Reaction Rates inSpheroidal Geometry, Application to Repressor-Operator Association andMembrane Bound Enzymes, Biophysical Chemistry (1974), 2; 255-263.

10. Nygren, H., Werthen, M., & Stenberg, M., Kinetics of AntibodyBinding to Solid-Phase-Immobilized Antigen, J. Immunol. Meth. (1987),101; 63-71.

11. Barbas, C. F. III et al. Assembly of Combinatorial Libraries onPhage Surfaces: The Gene III Site. Proc. Natl. Acad, Sci. USA (1991),88; 7978-7982.

12. Dixon, M. & Webb E. C. Enzymes (1979) Academic Press, New York, pp.169-181.

13. Goodenough, U. Genetics (2d Ed. 1978) Saunders College,Philadelphia, pp. 1578-68.

14. Shokat, K. M. and Schultz, P. G., Catalytic Antibodies (1991) Wiley,N.Y., pp 118-34.

15. A. Ferscht, Enzyme Structure and Mechanism, 2nd Ed., pp. 101-103, W.H. Freeman & Co. NY (1985).

16. Tang et al., Proc. Natl. Acad. Sci. (1991), 88; 8784-86.

17. Inman and Barnett (1989), “Protein Recognition of ImmobilizedLigands,” T. W. Hutchens Ed., A. R. Liss, NY pp. 35-44).

18. Marks et al., By-Passing Immunization. Human antibodies form V-geneLibraries Display on Phage, J. Mol. Biol. (1991), 222; 581-97.

19. Hoogenboom et al., Multi-subunit Proteins on the Surface ofFilamentous Phage: Methodologies for Displaying Antibody (Fab) Heavy andLight Chains, Nucl. Acids Res. (1991) 19; 4133-37.

20. Chang et al., Expression of Antibody Fab Domains on BacteriophageSurfaces, J. Immunol. (1991) 147; 3610-14.

21. Ostrove, S. in Methods in Enzymology, In, (M. P. Deutscher, ed.)1990, Academic Press, New York, pp 357-371.

22. Sundarum, P. V. & Eckstein, F. Theory and Practice in AffinityTechniques, 1978, Academic Press, New York.

23. Morgan, M. R. A. & Dean, P. D. G. in Theory and Practice in AffinityTechniques (Sundarum, P. V. & Eckstein, F., eds.) 1978, Academic Press,New York, pp 14-22.

24. Stellwagon, E. in Methods in Enzymology, 1, (M. P. Deutscher, ed.)1990, Academic Press, New York, pp 343-357.

25. Ostrove, S. & Weiss, S. in Methods in Enzymology, In, (M. P.Deutscher, ed.) 1990, Academic Press, New York, pp 371-379.

26. Haff, L. A. & Easterday, R. L. in Theory and Practice in AffinityTechniques (Sundarum, P. V. & Eckstein, F., eds.) 1978, Academic Press,New York, pp 24-44.

27. Smith, M. C., Furman, T. C., Ingolia, T. D., & Pidgeon, C. J. Biol.Chem. 263, (1988) pp 7211-7215.

28. Carlsson, J., Svenson, A., Ryden, L. in Solid Phase Methods inProtein Sequencing Analysis, (Previero, A. & Coletti-Previero, M. -A.,ed.) 1977, Elsevier/North Holland Biomedical Press, pp 29-37.

29. Bonnafous, J. C., Dornand, J., Faver, J., Sizes, M., Boschetti, E.,Mani, J. C., J. Immunol. Methods 58, (1988) pp 93-107.

30. Pierce ImmunoTechnology Catalog & Handbook, (1991), pp D-12-D-13,and references therein.

31. Skerra, A., et al., “Filter Selection of Antibody Fab FragmentsSecreted From Individual Bacterial Colonies:

Specific Detection of Antigen Binding With A Two-Membrane System”Analytical Biochemistry (1991), 1; 151-55.

32. McCafferty, J. et al., “Phage-Enzymes: Expressing And AffinityChromatography Of Functional Alkaline Phosphatase On The Surface OfBacteriophage,” Protein Engineering, (1991), vol. 4, no. 8, pp 955-61.

33. Comprehensive Organic Chemistry D. Barton & W. D. Ollis eds., Vol.1-6, Pergamon Press, NY (1979).

Having thus described in detail the preferred embodiments of the presentinvention, it is to be understood that the invention defined by theappended claims is not to be limited by particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention.

4 22 base pairs nucleic acid single linear DNA (genomic) unknown 1AGGTGAAACT GCAGGAGTCA GG 22 32 base pairs nucleic acid single linear DNA(genomic) unknown 2 TGAGGAGACG GTGACCGTGG TCCCTTGGCC CC 32 24 base pairsnucleic acid single linear DNA (genomic) unknown 3 GACATTGAGC TCACCCAGTCTCCA 24 97 base pairs nucleic acid single linear DNA (genomic) unknown 4CCGTTTGATT TCCAGCTTGG TGCCCCGTTT TATTTTCCAG CTTGGTCCCC CGTTTTATTT 60CCAACTTTGT CCCCCGTTTC AGCTCCAGCT TGGTCCC 97

What is claimed is:
 1. A method for producing a recombinant virus or acell-line capable of expression of a surface-bound catalytic moietycomprising: selection for binding to a selected molecule a population ofrecombinant viruses suspected of including viruses expressing thecatalytic moiety, isolating from said population-a first subpopulationwhich can bind to the selected molecule, reaction-based selection ofsaid first subpopulation for catalytic activity by a surface-boundcatalytic moiety, isolating from said first subpopulation a secondsubpopulation which can act catalytically, and, infecting a hostsusceptible to infection by the recombinant virus with virus of thesecond subpopulation.
 2. The method of claim 1 wherein the selection forcatalytic activity comprises contacting said first subpopulation with amechanism-based inhibitor so as to form a reaction mixture.
 3. Themethod of claim 2 wherein the selection of the population for binding tothe selected molecule comprises (i) contacting the population with amedium containing at least one immobilized substrate or hapten, (ii)isolating the first subpopulation by collecting that portion of thepopulation which binds to the substrate or hapten, and (iii) isolatingthe second subpopulation by contacting the reaction mixture with amedium containing immobilized substrate or hapten and collecting viruseswhich do not bind to the immobilized substrate or hapten.
 4. The methodof claim 3 wherein the medium containing immobilized substrate or haptenin either or both of the selection and isolating steps comprises anaffinity column of immobilized substrate or hapten.
 5. The method ofclaim 1 wherein the selection for catalytic activity comprisescontacting said first subpopulation with a surface including a desiredsubstrate of a catalytic reaction, said contacting being at a firstpoint on said surface, and allowing sufficient time to pass for membersof said first subpopulation having catalytic activity to move a distanceto a second point on the surface.
 6. The method of claim 5 wherein theselection of the population for binding to the desired substratecomprises (i) passing the population over an affinity column comprisingimmobilized hapten and eluting that portion of the population whichbinds to the affinity column to isolate said first subpopulation, and(ii) collecting the members of the first subpopulation from the secondpoint on the surface to isolate the second subpopulation.
 7. The methodof claim 1 wherein the selection for catalytic activity comprises (i)contacting said first subpopulation with a surface including a desiredsubstrate of a catalytic reaction to which members of the firstsubpopulation not having catalytic activity will bind and members of thefirst subpopulation having catalytic activity will bind and engage incatalysis, and after sufficient time for members of said firstsubpopulation not having catalytic activity to approach equilibrium withthe surface but less than the time from contacting for members of saidfirst subpopulation having catalytic activity to consume said substrate,(ii) washing said surface to remove therefrom members of the firstsubpopulation having catalytic activity; and (iii) isolating the secondsubpopulation by collecting the wash from washing step (ii).
 8. Themethod of claim 7 wherein the selection of the population for binding tothe desired substrate comprises passing the population over an affinitycolumn comprising immobilized hapten and eluting that portion of thepopulation which binds to the affinity column to isolate the firstsubpopulation.
 9. The method of claim 7 wherein the selection forcatalytic activity further comprises, after contacting said firstsubpopulation with the surface, prior to washing the surface to removetherefrom members of the first subpopulation having catalytic activityand after sufficient time from the contacting for both members of thefirst subpopulation not having catalytic activity and members of thefirst subpopulation having catalytic activity to bind to the substratebut less than the time from contacting for members of said firstsubpopulation having catalytic activity to complete a catalyticreaction, washing said surface so as to remove any members of said firstsubpopulation having low or no affinity to the substrate.
 10. The methodof claim 1 wherein the selection for catalytic activity comprises (i)contacting the first subpopulation with substrate at a first temperatureat which binding but substantially no catalysis will occur, and thencontacting the first population with substrate at a second temperaturewhich is higher than the first temperature, and at which both bindingand catalysis will occur; and (ii) isolating the second subpopulation bycollecting those members of the first subpopulation in which temperatureaffects apparent binding.
 11. The method of claim 1 wherein theselection for catalytic activity comprises passing the populationthrough a medium having mechanism-based inhibitor bound thereto by acleavable group, and isolating the second subpopulation comprisescollecting those members of the first subpopulation bound to theinhibitor by cleaving the inhibitor from the medium.
 12. The method ofclaim 1 wherein the selection for catalytic activity comprises (i)contacting the first subpopulation with an immobilized non-reactivesubstrate analog in the presence of mobile non-reactive substrate analogand collecting therefrom a plurality of samples which bind to theimmobilized non-reactive substrate analog with different bindingcharacteristics, and then contacting the samples with the immobilizednon-reactive substrate analog in the presence of mobile substrate; and(ii) isolating the second subpopulation by collecting those members ofeach of the samples of the first subpopulation which exhibit differentapparent binding than a majority of the members in each of the samplesof the first subpopulation.
 13. The method of claim 1 wherein theselection for catalytic activity comprises contacting the firstsubpopulation with a mechanism based inhibitor bound to mobile particlesby a cleavable group, and isolating the second subpopulation comprisesseparating the particles from the first subpopulation and collectingthose members thereof bound to the inhibitor by cleaving the inhibitorfrom the particles.
 14. The method of claim 13 wherein the particles areseparated by filtration.
 15. The method of claim 13 wherein theparticles are separated by gravity or centrifugal force.
 16. The methodof claim 13 wherein the particles are magnetically responsive and areseparated by imposing a magnetic field.
 17. The method of claim 5wherein movement of members of the first subpopulation having catalyticactivity is enhanced by applying an electric field to the surface. 18.The method of claim 1 wherein the recombinant virus is an fd or M13phage, and the catalytic moiety is a catalytic antibody which is capableof catalyzing an ester hydrolysis, or is a catalytic portion thereof.19. The method of claim 2 wherein the recombinant virus is an fd or M13phage, and the catalytic moiety is a catalytic antibody which is capableof catalyzing an ester hydrolysis, or is a catalytic portion thereof.20. The method of claim 5 wherein the recombinant virus is an fd or M13phage, and the catalytic moiety is a catalytic antibody which is capableof catalyzing an ester hydrolysis or is a catalytic portion thereof. 21.The method of claim 7 wherein the recombinant virus is an fd or M13phage, and the catalytic moiety is a catalytic antibody which is capableof catalyzing an ester hydrolysis or is a catalytic portion thereof. 22.The method of claim 10 wherein the recombinant virus is an fd or M13phage, and the catalytic moiety is a catalytic antibody which is capableof catalyzing an ester hydrolysis or is a catalytic portion thereof. 23.The method of claim 11 wherein the recombinant virus is an fd or M13phage, and the catalytic moiety is a catalytic antibody which is capableof catalyzing an ester hydrolysis or is a catalytic portion thereof. 24.The method of claim 12 wherein the recombinant virus is an fd or M13phage, and the catalytic moiety is a catalytic antibody which is capableof catalyzing an ester hydrolysis or is a catalytic portion thereof. 25.The method of claim 13 wherein the recombinant virus is an fd or M13phage, and the catalytic moiety is a catalytic antibody which is capableof catalyzing an ester hydrolysis, or a catalytic portion thereof. 26.The method of claim 1 wherein the recombinant virus is an fd or M13phage and host is E. coli.
 27. The method of claim 1 wherein thecell-line is an E. coli cell-line.
 28. A substantially pure recombinantvirus population which expresses a catalytic moiety which is a catalyticantibody or catalytic portion thereof produced by the method of claim 1.29. A substantially pure cell-line which expresses a catalytic moietywhich is a catalytic antibody or catalytic portion thereof, produced bythe method of claim
 1. 30. A substantially pure recombinant viruspopulation which expresses a catalytic moiety which is a catalyticantibody or catalytic portion thereof produced by the method of claim 2.31. A substantially pure recombinant virus population which expresses acatalytic moiety which is a catalytic antibody or catalytic portionthereof produced by the method of claim
 5. 32. A substantially purerecombinant virus population which expresses a catalytic moiety which isa catalytic antibody or catalytic portion thereof produced by the methodof claim
 7. 33. A substantially pure recombinant virus population whichexpresses a catalytic moiety which is a catalytic antibody or catalyticportion thereof produced by the method of claim
 10. 34. A substantiallypure recombinant virus population which expresses a catalytic moietywhich is a catalytic antibody or catalytic portion thereof produced bythe method of claim
 11. 35. A substantially pure recombinant viruspopulation which expresses a catalytic moiety which is a catalyticantibody or catalytic portion thereof produced by the method of claim12.
 36. A substantially pure recombinant virus population whichexpresses a catalytic moiety which is a catalytic antibody or catalyticportion thereof produced by the method of claim
 13. 37. A substantiallypure recombinant fd or M13 phage population produced by the method ofclaim
 18. 38. A substantially pure recombinant fd or M13 phagepopulation produced by the method of claim
 19. 39. A substantially purerecombinant fd or M13 phage population produced by the method of claim20.
 40. A substantially pure recombinant fd or M13 phage populationproduced by the method of claim
 21. 41. A substantially pure recombinantfd or M13 phage population produced by the method of claim
 22. 42. Asubstantially pure recombinant fd or M13 phage population produced bythe method of claim
 23. 43. A substantially pure recombinant fd or M13phage population produced by the method of claim
 24. 44. A substantiallypure recombinant fd or M13 phage population produced by the method ofclaim 25.